Figure 3. Gal 3 specifically enhances LPS-mediated neutrophil activation and requires a direct interaction with LPS for its effect.

A: PMNs were primed for 30 min at 37°C with Gal 3 (0.2 µM) or E. coli LPS (1 µg/mL) and then stimulated with LPS (1 µg/mL) or Gal 3 (0.2 µM), respectively. Alternatively, Gal 3 (1 µM) and LPS (5 µg/mL) were preincubated together for 30 min at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs (n = 2). B: Effect of increasing LPS and Gal 3 concentrations in the pre-incubation mixture. Increasing concentrations of Gal 3 (0.2 µM to 25 µM) and LPS (1 µg/mL to 125 µg/mL) were preincubated together for 30 min at 37°C. They were then diluted in HBSS²⁺ to the final Gal 3 − 0.2 µM and LPS – 1 µg/ml concentrations and added to PMN for a 45-min incubation at 37°C. C: Time-response curve of LPS and Gal 3 preincubation. Gal 3 (7 µM) and LPS (35 µg/mL) were preincubated together for 0–30 min, as indicated, at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs for a 45-min incubation at 37°C (representative from 2 independent experiments). D: PMNs were stimulated with LPS (1 µg/ml), fMLP (10⁻⁶ M), TNF-α (10 ng/ml) or IL-8 (25 ng/ml), which had all been preincubated without (black) or with Gal 3 (0.4 µM, grey) for 30 min at 37°C. Neutrophil CD11b expression was measured as in figure 1 (n = 5). E: Gal 3 effect on neutrophil activation by LPS from various strains of bacteria. Gal 3 (1 µM) was preincubated with 10 µg/ml LPS from E. coli, K. pneumonia or S. minnesota R7 for 30 min at 37°C, in presence or absence of 10 mM lactose (lac). Then, each mixture was diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) concentrations and used to stimulate PMNs (2×10⁶/mL) for 45 min at 37°C, and analyzed for CD11b expression as above. Results are expressed as mean fluorescence intensity (MFI) (n = 3). * p<0.05, **p<0.01, ***p<0.001.