Figure 12.

SLPI signaling involves annexin 2 (Anx2). Expression of Anx2 in U937 cells was diminished by siRNA. Cells were primed with LPS (1 μg/ml) for 5 h and further stimulated with 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt (BzATP; 100 mM) for 30 min in the presence or absence of SLPI (10 μg/ml). The efficiency and specificity of the knock-down was confirmed by immunoblotting. The concentration of IL-1β in the cell culture supernatant was measured by ELISA. (A) The protein expression level of Anx2 was silenced upon transfection with Anx2 siRNA; one representative immunoblot out of 4. (B) The optical density of the immuno-positive bands was measured and divided by the values obtained for β-actin on the same blot. The values gathered from untreated cells were set to one and all other values were calculated accordingly. ^*p ≤ 0.05 significantly different compared to cells transfected with control (con) siRNA. (C) Down-regulation of the expression of Anx2 siRNA blunted the SLPI-induced inhibitory effect. ^*p ≤ 0.05 significantly different compared to cells transfected with control siRNA (con) and stimulated with LPS, BzATP and SLPI. (D,E) Conditioned media were collected from U937 cells transfected with control siRNA (con) (M3), Anx2 siRNA (M4), or with iPLA2β siRNA (M5). All cells were primed before media collection with LPS for 5 h and SLPI was applied for additional 30 min. The low molecular mass fractions (LMMF) of M4 and M5 were devoid of inhibitory activity. ^*p ≤ 0.05 significantly different compared to U937 cells treated with LMMF of M3. Experimental groups were compared by Kruskal-Wallis test followed by Mann-Whitney rank sum test.