Chemokines (CCL3, CCL4, and CCL5) Inhibit ATP-Induced Release of IL-1β by Monocytic Cells

Figure 6

CCL3 signaling involves calcium-independent phospholipase A2 (iPLA2). Human monocytic U937 cells were primed with LPS (1 μg/ml, 5 h) and activated 2′(3′)-O-(4-Benzoylbenzoyl)adenosine-5′-triphosphate (BzATP; 100 μM, 30 min). The release of IL-1β to the cell culture supernatant was measured by ELISA. (a) CCL3 (10 ng/ml) inhibited the release of IL-1β in response to BzATP. The general PLA2 inhibitor arachidonyl trifluoromethyl ketone (ATK) and the specific iPLA2 inhibitor bromoenol lactone (BEL) reversed CCL3-dependent inhibition. (b–e) Expression of iPLA2 by U937 cells was silenced by siRNA (PLA2G6); scrambled siRNA served as control. Silencing of PLA2G6 expression was efficient as revealed by real-time RT-PCR (b) and by Western blotting (c, d). Data in (b) and (d) are given as arbitrary units. (c, d) In Western blotting experiments, β-actin served as a loading control, the optical density (OD) of the immunopositive bands was measured, and the ratio of the OD of iPLA2 and β-actin was formed. (b, d) Values obtained for cells treated with siRNA targeting CCR1 were statistically compared to those transfected with control siRNA. (e) After treatment with siRNA targeting PLA2G6 expression, the inhibitory effect of CCL3 on IL-1β release was blunted. In untreated control cells and upon transfection of control siRNA, CCL3 was effective. Data are presented as individual data points, bars indicate median, and whiskers percentiles are 25 and 75, , each.

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