Figure 2 - Rat macrophage galectin induces apoptosis of T cells. A, Electrophoretic analysis of internucleosomal DNA fragmentation induced by RMGal. Spleen mononuclear cells were cultured in 24-well microtiter plates at a density of 2 x 107 cells/well for 6 h in medium alone (lane 1), in medium containing Con A, 2.5 g/ml (lane 2), and in medium containing Con A, 2.5 g/ml, plus the addition of RMGal at a concentration of 4 g/ml (lane 3). Cells were also cultured with RMGal (4 and 6 g/ml) in the absence of a mitogenic stimulus (lanes 4 and 5, respectively). The T cell-enriched population was purified and cultured under identical conditions in medium alone (lane 6), in medium containing Con A (lane 7), in the presence of Con A plus RMGal, 4 g/ml (lane 8), and in the presence of RMGal alone, 4 g/ml (lane 9). Cells were then harvested and genomic DNA was extracted. Samples were diluted in loading buffer and resolved on 1.8% agarose gel. The relative mobility of oligonucleosome-length DNA fragments reflects integer multiples of ~180-200 bp. Molecular standards (100 bp DNA ladder) are indicated on the right. B, Incorporation of biotinylated dUPT by exogenous TdT into DNA strand breaks generated after RMGal treatment. T cells (2 x 107 cells/well) were exposed to medium alone (a), 4 g/ml RMGal (b), 2.5 g/ml Con A (c), 2.5 g/ml Con A plus 4 g/ml RMGal (d), 4 g/ml RMGal in the presence of 100 mM lactose (e), or 4 g/ml RMGal in the presence of galectin Ab (f) for 6 h at 37oC in 5% CO2. Samples were harvested, fixed, and permeabilized and the percentage of apoptotic cells in each sample was determined by flow cytometry analysis after TUNEL labeling. Cells treated with DNAse I were used as positive controls. C, Transmission electron microscopy examination of ultrastructural changes induced by RMGal. T cells were purified and cultured in 24-well plates at a density of 2 x 107 cells/well, in the presence (1), or in the absence (2) of RMGal (4 g/ml). After 6 h, cells were harvested, washed and processed for transmission electron microscopy. Galectin-treated cells displayed the typical ultrastructural features compatible with apoptosis. Magnification: X12,000. (From Journal of Immunology, 160: 4831-4840, 1998. Copyright 1998, The American Society of Immunologists).