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

Figure 3

Signaling via chemokine receptor CCR1. (a) The mRNA expression of chemokine receptors CCR1, CCR3, CCR5, and CXCR6 was investigated in U937 cells by real-time RT-PCR, and the amplicons were separated by gel electrophoreses together with a base pair (bp) ladder and detected by GelRed™ Nucleic Acid Dye. Peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers served as positive control. (b, c) U937 cells were transfected with scrambled control siRNA or with siRNA targeting CCR1. These cells were primed with LPS (1 μg/ml, 5 h) followed by activation with 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate (BzATP) (100 μM, 30 min). (b) The efficiency of the siRNA transfection was verified by real-time RT-PCR. Values obtained for cells treated with siRNA targeting CCR1 were statistically compared to those transfected with control siRNA. (c) IL-1β was measured in the cell culture supernatant by ELISA. In untreated and in control-transfected cells, CCL3 (10 ng/ml) fully inhibited the BzATP-induced release of IL-1β, whereas silencing of CCR1 significantly blunted the inhibitory effect of CCL3. In all experiments, acetylcholine (ACh; 10 μM) was included as a positive control. Data are presented as individual data points, bars indicate median, and whiskers percentiles are 25 and 75; .

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