Effects of neutralizing antibodies against various adhesion molecules and chemokines on the transmigration times of neutrophils, PBLs, and monocytes and their randomness in movements beneath the EC monolayers in the reattachment flow area c under VSF
. | Randomness, T/N . | . | . | Transmigration time, sec . | . | . | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| mAb . | Neutrophils . | PBLs . | Monocytes . | Neutrophils . | PBLs . | Monocytes . | ||||
| NA | 10.88 ± 1.92 | 1.52 ± 0.25 | 1.12 ± 0.02 | 58.27 ± 2.13 | 131.21 ± 8.11 | 204.40 ± 7.44 | ||||
| IgG | 9.73 ± 1.74 | 1.42 ± 0.23 | 1.16 ± 0.02 | 63.28 ± 6.75 | 141.32 ± 6.36 | 195.32 ± 8.22 | ||||
| ICAM-1 | 6.89 ± 1.12* | 1.46 ± 0.17 | 1.12 ± 0.02 | 207.13 ± 9.57* | 189.27 ± 7.22* | 276.28 ± 9.46* | ||||
| VCAM-1 | 11.13 ± 2.17 | 1.04 ± 0.02* | 1.14 ± 0.03 | 65.23 ± 6.42 | 231.39 ± 9.40* | 218.19 ± 8.32 | ||||
| E-selectin | 9.61 ± 1.75 | 1.51 ± 0.31 | 1.11 ± 0.03 | 228.46 ± 11.72* | 125.53 ± 6.31 | 210.64 ± 8.16 | ||||
| MCP-1 | 13.53 ± 2.06 | 1.38 ± 0.17 | 1.10 ± 0.02 | 66.02 ± 8.10 | 204.61 ± 10.06* | 267.24 ± 10.34* | ||||
| IL-8 | 11.72 ± 1.52 | 1.37 ± 0.19 | 1.08 ± 0.03 | 243.50 ± 10.22* | 146.45 ± 8.42 | 214.65 ± 8.75 | ||||
| RANTES | 14.98 ± 1.68 | 1.38 ± 0.14 | 1.13 ± 0.02 | 67.33 ± 7.35 | 148.31 ± 8.20 | 211.51 ± 8.29 | ||||
| GRO-α | 11.14 ± 2.12 | 1.44 ± 0.17 | 1.10 ± 0.03 | 106.43 ± 6.34* | 138.48 ± 7.26 | 217.40 ± 8.40 | ||||
| IP-10 | 11.49 ± 1.66 | 1.42 ± 0.14 | 1.11 ± 0.02 | 65.19 ± 9.61 | 151.32 ± 7.46 | 214.67 ± 9.92 | ||||
| I-TAC | 13.91 ± 2.57 | 1.49 ± 0.11 | 1.12 ± 0.03 | 70.11 ± 8.50 | 144.54 ± 6.17 | 221.74 ± 7.64 | ||||
| SDF-1 | 10.77 ± 1.84 | 1.42 ± 0.12 | 1.11 ± 0.02 | 62.56 ± 6.68 | 168.51 ± 7.74* | 223.71 ± 8.26 | ||||
. | Randomness, T/N . | . | . | Transmigration time, sec . | . | . | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| mAb . | Neutrophils . | PBLs . | Monocytes . | Neutrophils . | PBLs . | Monocytes . | ||||
| NA | 10.88 ± 1.92 | 1.52 ± 0.25 | 1.12 ± 0.02 | 58.27 ± 2.13 | 131.21 ± 8.11 | 204.40 ± 7.44 | ||||
| IgG | 9.73 ± 1.74 | 1.42 ± 0.23 | 1.16 ± 0.02 | 63.28 ± 6.75 | 141.32 ± 6.36 | 195.32 ± 8.22 | ||||
| ICAM-1 | 6.89 ± 1.12* | 1.46 ± 0.17 | 1.12 ± 0.02 | 207.13 ± 9.57* | 189.27 ± 7.22* | 276.28 ± 9.46* | ||||
| VCAM-1 | 11.13 ± 2.17 | 1.04 ± 0.02* | 1.14 ± 0.03 | 65.23 ± 6.42 | 231.39 ± 9.40* | 218.19 ± 8.32 | ||||
| E-selectin | 9.61 ± 1.75 | 1.51 ± 0.31 | 1.11 ± 0.03 | 228.46 ± 11.72* | 125.53 ± 6.31 | 210.64 ± 8.16 | ||||
| MCP-1 | 13.53 ± 2.06 | 1.38 ± 0.17 | 1.10 ± 0.02 | 66.02 ± 8.10 | 204.61 ± 10.06* | 267.24 ± 10.34* | ||||
| IL-8 | 11.72 ± 1.52 | 1.37 ± 0.19 | 1.08 ± 0.03 | 243.50 ± 10.22* | 146.45 ± 8.42 | 214.65 ± 8.75 | ||||
| RANTES | 14.98 ± 1.68 | 1.38 ± 0.14 | 1.13 ± 0.02 | 67.33 ± 7.35 | 148.31 ± 8.20 | 211.51 ± 8.29 | ||||
| GRO-α | 11.14 ± 2.12 | 1.44 ± 0.17 | 1.10 ± 0.03 | 106.43 ± 6.34* | 138.48 ± 7.26 | 217.40 ± 8.40 | ||||
| IP-10 | 11.49 ± 1.66 | 1.42 ± 0.14 | 1.11 ± 0.02 | 65.19 ± 9.61 | 151.32 ± 7.46 | 214.67 ± 9.92 | ||||
| I-TAC | 13.91 ± 2.57 | 1.49 ± 0.11 | 1.12 ± 0.03 | 70.11 ± 8.50 | 144.54 ± 6.17 | 221.74 ± 7.64 | ||||
| SDF-1 | 10.77 ± 1.84 | 1.42 ± 0.12 | 1.11 ± 0.02 | 62.56 ± 6.68 | 168.51 ± 7.74* | 223.71 ± 8.26 | ||||
The EC/SMC cocultures were incubated with neutralizing antibodies (20 μg/mL) against the indicated adhesion molecules and chemokines for 1 hour prior to adhesion and transmigration assays under VSF. The EC/SMC treated with isotype-matched IgG or without antibody treatment (NA) was used as controls. Purified neutrophils, PBLs, or monocytes were perfused over the EC/SMC under VSF for 20 minutes, and video records were made of the behaviors of adhesion and transmigration in the reattachment flow area c. The transmigration times of purified WBCs as well as the total distances (T), net distances (N), and randomness (T/N) of their subendothelial migration were measured by computerized image analysis, as described in “Materials and methods.” An example is provided in Video S6 to show an increase in transmigration time of neutrophils by the use of antibody to ICAM-1 as compared with the control IgG. Data shown in this table are mean ± SEM of 25 cells from three independent experiments. A complete list of the results on all parameters for all areas under VSF and static condition is provided in Table S2.
P < .05 versus control EC/SMC treated with IgG or without antibody treatment