Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation

Abstract

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.

Fig. 1.

Lectin binding following HUVEC oxidative stress (ELISA). The effect of endothelial oxidative stress on lectin (10 μg/mL) binding was investigated by ELISA. Only UEA-II deposition following endothelial oxidative stress was significantly increased compared to normoxic cells. Data are presented as the percent change in lectin binding compared to normoxia with the data being normalized to the measured optical density (O.D.) of each respective lectin at baseline (normoxia). (n = 3; error bars = SEM; *P < 0.05 compared to normoxic cells; see Table 1 for lectin abbreviations).

Fig. 2.

Competitive inhibition of MBL or UEA-II binding following HUVEC oxidative stress (ELISA). Competitive binding ELISA were performed in order to determine whether MBL and UEA-II compete for a common endothelial binding site following oxidative stress. (A) Treatment of HUVEC with UEA-II significantly (P < 0.05) attenuated MBL binding in a concentration-dependent manner following oxidative stress. (B) Addition of increasing amounts of purified human MBL significantly (P < 0.05) attenuated UEA-II binding to HUVEC following oxidative stress. (n = 3; error bars = SEM; *P < 0.05 compared to vehicle).

Fig. 3.

UEA-II attenuates C3 deposition following endothelial oxidative stress (ELISA). C3 deposition following endothelial oxidative stress was measured by ELISA. C3 deposition following endothelial oxidative stress was significantly decreased in a concentration-dependent manner by treatment of HUVEC with UEA-II (IC₅₀ ≈ 10 pmol/L). Data are normalized to the measured O.D. of untreated hypoxic/reoxygenated cells (O.D. at 450 nm = 0.77 ± 0.12) and expressed as percent inhibition. (n = 6, error bars = SEM, *P < 0.05 compared to vehicle).

Fig. 4.

UEA-II attenuates MBL and C3 deposition following endothelial oxidative stress (confocal microscopy). Confocal microscopical demonstration of HUVEC MBL (blue) and C3 (green) deposition following oxidative stress. Treatment of HUVEC with UEA-II (B) 0.1 fmol/L; (C) 100 fmol/L; (D) 100 pmol/L; (E) 100 nmol/L UEA-II) decreased MBL and C3 deposition and co-localization (white) in a concentration-dependent manner compared to untreated cells (A) following oxidative stress. This figure is representative of three experiments. (Endothelial nuclei are indicated by red.)

Fig. 5.

UEA-II does not alter serum hemolytic activity. Complement hemolytic assays using sensitized chicken red blood cells were performed in order to demonstrate that UEA-II does not directly inhibit or activate complement. Treatment of HS with UEA-II (1 μmol/L) did not significantly alter hemolytic activity compared to untreated HS. (n = 2, error bars = SEM.)

Fig. 6.

Neutrophil chemotaxis is attenuated following oxidative stress by treatment of HUVEC with UEA-II. Oxidative stress significantly increased neutrophil chemotaxis compared to normoxic cells. Treatment of HUVEC with UEA-II (100 nmol/L) significantly attenuated neutrophil chemotaxis following endothelial oxidative stress. Neutrophil chemotaxis to HUVEC following oxidative stress was measured by analysis of myeloperoxidase levels and transformed to neutrophil count with a standard curve (n = 3, error bars = SEM, *P < 0.05 compared to normoxia, P < 0.05 compared to vehicle-treated hypoxia/reoxygenation cells).