In addition, production of mucus by goblet cells was also diminished. of the peptides was able to induce a chemotactic response in THP-1 cells. Number 1a shows a representative experiment using 042 M of CDP-1 and CDIP-2 peptides, and the related CCL13 control used at 01 M (which corresponds to the maximum of doseCresponse in chemotaxis for THP-1 cells, data not demonstrated). To characterize further the potential chemotactic response to CDPs, we tested different concentrations of these peptides (0004C42 M). In Fig. 1b representative data are demonstrated of the chemotactic response of CDIP-2. None of these concentrations induced a significant chemotactic response on THP-1 cells. In addition, to determine whether these peptides were capable of inducing intracellular calcium fluxes, different peptide concentrations (0C42 M) were used to stimulate THP-1 cells and calcium responses were measured using a circulation cytometer (Fig. 1c). As demonstrated, both peptides, CDP-1 and CDIP-2 (at 42 M) were incapable of inducing calcium flux reactions in THP-1 cells compared with CCL13 (01 M), which induced significant calcium mobilization. Open in a separate windowpane Fig. 1 Analysis of the agonistic properties of chemokine (C-C motif) ligand 13 (CCL13)-derived peptides (CDPs) in tumour human being peripheral blood monocytic (THP-1) cells. (a) Chemotaxis assays. THP-1 monocytic cells were used in a chemotaxis assay using the CDPs as agonists, as explained in Methods. Peptides were used at a concentration of 042 M. The chemokine CCL13 (01 M) was used as control. Data are indicated as the mean migration index as explained in Methods. (b) DoseCresponse analysis of CDIP-2 ADAMTS9 chemotaxis assays using different concentrations of CDIP-2 peptide (0004C42 M). CCL13 (01 M) was used as control. (c) Calcium flux assays. Calcium flux SCR7 pyrazine reactions induced by CDPs was monitored by cytometry and displayed as relative fluorescence percentage. THP-1 cells were stimulated with 84 M of CDPs and 01 M of the chemokine CCL13 at different time-points (arrow). Tracings are from a representative three self-employed experiments. Analysis of the antagonistic activities by CCL13-derived peptides Because no chemotactic reactions were induced by CDPs, we explored SCR7 pyrazine the possibility that these peptides could be acting as antagonists, probably by binding the chemokine receptors without inducing activation or by obstructing the binding of CCL13 to their receptors. First, to evaluate their antagonistic effect, THP-1 cells were preincubated with different concentrations of each peptide (1C42 M) for 15 min at 4C, prior to chemotaxis assays using CCL13 (1 M) as agonist. Interestingly, CDIP-2 ( 21 M) induced more than 40% of inhibition of the chemotactic response of THP-1 cells compared with additional CDP. At lesser concentrations (1 M), it experienced little or no effect on CCL13-mediated chemotaxis (Fig. 2a). In addition, we SCR7 pyrazine identified the effect of CDIP-2 on both human SCR7 pyrazine being peripheral blood mononuclear cells and murine peritoneal mononuclear cells, showing a similar antagonistic effect (data not demonstrated). Furthermore, we identified whether the CDPs might have an inhibitory effect on the calcium flux response. Peptides (42C84 M) were added to monocytic cells before adding CCL13 (01 M) (Fig. 2b) and SCR7 pyrazine calcium fluxes were determined by FACS analysis. As demonstrated, none of the tested peptides (CDIP-2 and CDP-1) inhibited the calcium flux induced by CCL13. Open in a separate windowpane Fig. 2 Antagonistic activities induced by chemokine (C-C motif) ligand 13 (CCL13)-derived peptides (CDPs) in tumour human being peripheral blood monocytic (THP-1) cells. (a) CDIP-2 peptide inhibits CCL13-mediated chemotactic reactions of THP-1 cells. Cells were preincubated with different concentrations of either peptide CDIP-2 (1C42 M) or CCL13 (1 M) previous to the chemotaxis assay using CCL13 (1 M) like a chemoattractant. Results are demonstrated as percentage of inhibition standard error (* 001). (b) Analysis of the antagonist.