Internalization of α-defensin by SMCs.

(A) Human umbilical vein SMCs were incubated for 1 hour at 4°C with 2 μM ¹²⁵I-α-defensin. Washing of the cells terminated incubation. Bound ¹²⁵I-defensin released immediately from the surface of SMCs (a) or after subsequent warming to 37°C for 30 minutes (b2). Internalized ¹²⁵I-α-defensin at 4°C (c) or 37°C (d). (B) The role of LRP in binding and internalization of α-defensin by SMCs. Experiments were performed as in panel A. Binding (a,b,c) and internalization (d,e,f) of¹²⁵I-defensin were determined at 37°C in the absence of additives (a,d) or in the presence of 20 nM anti-LRP antibody (b,e) or 20 nM rRAP (c,f). (C) Confocal fluorescence images of defensin binding and internalization by SMCs. Alexo 488–labeled antigens are depicted in red, and DIC images are shown in green. (i) SMCs incubated with antidefensin IgG (rabbit), followed by labeled goat antirabbit IgG, in the absence of α-defensin. (ii) SMCs incubated with 1 μM α-defensin for 30 minutes and later treated as in panel i. (iii) Cells preincubated for 10 minutes with anti-LRP antibodies (20 nM) before addition of 1 μM α-defensin and later treated as in panel B. (iv) Image reconstruction of 16 single sections presented in the color-coding scale. The upper section is shown in red, and the bottom section is shown in blue. Original magnification × 400.