Effect of kinase- or lipase-specific modulating compounds on CCR2 and CCR5 down-modulation
| Compound (μM) . | Primary target . | Percentage of CCR2 downmod to CCL2 ± SD (n) . | Percentage of CCR2 downmod to LTA ± SD (n) . | Percentage of CCR5 downmod to CCL5 ± SD (n) . | Percentage of CCR5 downmod to LTA ± SD (n) . |
|---|---|---|---|---|---|
| None | N/A | 40.25 ± 16.68 (10) | 35.52 ± 10.08 (10) | 34.35 ± 13.32 (10) | 28.14 ± 11.91 (10) |
| LY294002 [1.0] | PI3K inhibitor | 47.18 ± 15.12 (4) | 32.93 ± 8.52 (4) | 33.50 ± 14.41 (5) | 39.70 ± 19.12 (5) |
| Wortmannin [0.1] | PI3K inhibitor | 55.53 ± 13.23 (4) | 25.15 ± 1.97 (4) | 43.26 ± 19.18 (5) | 25.02 ± 17.69 (5) |
| PD98059 [20.0] | MAPKK (MEK) inhibitor | 52.35 ± 13.68 (4) | 28.58 ± 10.04 (4) | 35.44 ± 15.66 (5) | 36.18 ± 19.48 (5) |
| SB203580 [10.0] | p38 MAPK inhibitor | 40.86 ± 19.61 (2) | 44.54 ± 16.97 (2) | 53.94 ± 0.67 (2) | 58.04 ± 3.41 (2) |
| SP600125 [10.0] | JNK inhibitor | 51.37 ± 11.72 (2) | 61.42 ± 4.76 (2) | 56.19 ± 0.89 (2) | 58.81 ± 0.54 (2) |
| GF109203X [5.0] | PKC inhibitor | 33.73 ± 25.24 (4) | 24.50 ± 8.83 (4) | 34.40 ± 16.08 (4) | 35.32 ± 10.70 (5) |
| PMA [0.1] | PKC activator | 43.73 ± 28.33 (6) | 7.63 ± 3.19 (4) | 49.45 ± 19.87 (7) | -4.44 ± 14.06 (5) |
| U73122 [10.0] | PI-PLC inhibitor | 32.90 ± 12.01 (4) | 8.72 ± 10.89 (4) | 7.45 ± 5.27 (4) | 2.15 ± 1.81 (4) |
| Compound (μM) . | Primary target . | Percentage of CCR2 downmod to CCL2 ± SD (n) . | Percentage of CCR2 downmod to LTA ± SD (n) . | Percentage of CCR5 downmod to CCL5 ± SD (n) . | Percentage of CCR5 downmod to LTA ± SD (n) . |
|---|---|---|---|---|---|
| None | N/A | 40.25 ± 16.68 (10) | 35.52 ± 10.08 (10) | 34.35 ± 13.32 (10) | 28.14 ± 11.91 (10) |
| LY294002 [1.0] | PI3K inhibitor | 47.18 ± 15.12 (4) | 32.93 ± 8.52 (4) | 33.50 ± 14.41 (5) | 39.70 ± 19.12 (5) |
| Wortmannin [0.1] | PI3K inhibitor | 55.53 ± 13.23 (4) | 25.15 ± 1.97 (4) | 43.26 ± 19.18 (5) | 25.02 ± 17.69 (5) |
| PD98059 [20.0] | MAPKK (MEK) inhibitor | 52.35 ± 13.68 (4) | 28.58 ± 10.04 (4) | 35.44 ± 15.66 (5) | 36.18 ± 19.48 (5) |
| SB203580 [10.0] | p38 MAPK inhibitor | 40.86 ± 19.61 (2) | 44.54 ± 16.97 (2) | 53.94 ± 0.67 (2) | 58.04 ± 3.41 (2) |
| SP600125 [10.0] | JNK inhibitor | 51.37 ± 11.72 (2) | 61.42 ± 4.76 (2) | 56.19 ± 0.89 (2) | 58.81 ± 0.54 (2) |
| GF109203X [5.0] | PKC inhibitor | 33.73 ± 25.24 (4) | 24.50 ± 8.83 (4) | 34.40 ± 16.08 (4) | 35.32 ± 10.70 (5) |
| PMA [0.1] | PKC activator | 43.73 ± 28.33 (6) | 7.63 ± 3.19 (4) | 49.45 ± 19.87 (7) | -4.44 ± 14.06 (5) |
| U73122 [10.0] | PI-PLC inhibitor | 32.90 ± 12.01 (4) | 8.72 ± 10.89 (4) | 7.45 ± 5.27 (4) | 2.15 ± 1.81 (4) |
Monocytes were pretreated with vehicle (DMSO or H2O) or signalling modulator for 30 minutes at 37°C before addition of 100nM CCL2, CCL5, 10μg/mL LTA or BM alone and a further incubation of 1 hour. Cell-surface levels of CCR2 or CCR5 were determined by flow cytometry as described in “Methods.” Results from N independent experiments performed in triplicate are expressed as the percentage of downmodulation ± SD.
Downmod indicates downmodulation; PI3K, phosphatidyl inositol-3 kinase; MAPKK, mitogen-activated protein kinase kinase; MEK, mitogen-activated protein/extracellular signal-related kinase kinase; and PI, phosphatidylinositol.