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

Figure 5

Release of small mediators in response to CCL3. U937 cells were primed with LPS (1 μg/ml, 5 h) and activated with 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. CCL3, which was included as a positive control, inhibited the BzATP-induced release of IL-1β. An ultrafiltrate (UF) was produced containing the low molecular mass fraction (<3 kDa) of the cell culture supernatant (S) of LPS-primed U937 cells treated with CCL3 (10 ng/ml) for 30 min. For the production of control UF, CCL3 was added to the cell-free supernatant of LPS-primed U937 cells shortly before ultrafiltration. (a) The UF and the high molecular mass fraction (HF) obtained by ultrafiltration were separated in a 15% SDS-polyacrylamide gel along with a molecular mass marker (M) followed by silver staining. The arrow is pointing to the band corresponding to CCL3. Proteins with higher molecular mass are bovine serum albumin (66.5 kDa) and its contaminations that were added to the CCL3 preparation for stabilization. One typical result out of 5 experiments is depicted. (b) Control UF had no effect on the BzATP-induced release of IL-1β by LPS-primed U937 cells, whereas UF significantly reduced the IL-1β release. The effect of the UF was sensitive to conotoxins ArIB [V11L,V16D] (200 nM) and RgIA4 (200 nM). Data are presented as individual data points, bars indicate median, and whiskers percentiles are 25 and 75.

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