Figure 3.

Interleukin-2 (IL-2) stimulates thrombospondin 1 (TSP-1) expression in T cells and motility through TSP-1 and low-density lipoprotein receptor-related protein 1 (LRP1). (a) Gel analysis (6% SDS–PAGE) showing immunoprecipitation of biosynthetically labelled TSP-1 from the birch antigen-specific T-cell clone AF24 ([³⁵S]methionine; 3-hr pulse) after culture under different conditions for 5 days. IL-2 and IL-4 were used at a concentration of 10 ng/ml. (b) Agarose gel electrophoresis showing RT-PCR-amplified TSP-1 and β-actin fragments using mRNA isolated from AF24 cells cultured for different times with IL-2 and IL-2 and anti-CD3 as templates. Fragments were visualized by ethidium bromide staining and UV illumination. (c) Western blotting of material from conditioned medium of unprecipitated material from AF24 cells cultured with IL-2 for different times and separated on a 6% SDS–PAGE gel. After different times the cells were cultured in AIM-V medium over night. It is evident that the synthesis of TSP-1 increases with time and is IL-2-dependent. (d, e) Interleukin-2 stimulates cell surface expression of TSP-1 whereas IL-4 rather inhibits TSP-1 expression but stimulates LRP1 expression. The cells were incubated in the presence of IL-2 (10 ng/ml) or IL-4 (20 ng/ml) for 30 min and the cell surface expression of TSP-1 and LRP1 was subsequently determined using immunoprecipitation of material from surface biotinylated cells (d) or quantitative immunocytochemistry (e). (f) Influence of IL-2 (10 ng/ml) and IL-4 (10 ng/ml) on T-cell migration into a collagen matrix. Motility was determined after 30 min. (g) Silencing of TSP-1 and LRP1 abrogates the stimulatory effect of IL-2 on T-cell motility. Migration into a collagen gel was determined after transfection with control siRNA, TSP-1 siRNA (Alternative 1), or LRP1 siRNA (Alternative 1). The number of cells at different depths was determined after 30 min. (a–e) One representative experiment of three to six independent experiments; (f) mean values of three independent experiments and (g) one representative experiment of two independent experiments.