Fig. 9
CX3CL1 prevents HPMo activation by chemokines required for monocyte homing. a Role of chemokines in HPMo transmigration promoted by angiogenic factors. Human monocytes were treated with PBS or pertussis toxin (PTX) at 1 µg/ml before applying them under flow to HUVECs prestimulated with TNF or TNF + VEGF-A. n = 3 experiments, data are mean ± s.d., two-way ANOVA with Holm-Sidak adjustment. b Kinetics of calcium flux in monocyte induced by sequential stimulations with chemokines analyzed by Fura2 QBT ratiometric assay. Black arrows indicate the time of chemokine addition. The chemokines were tested at 50 ng/ml. c Effect of blocking CX3CR1 on calcium flux induced by sequential stimulation of monocytes by chemokines. Monocytes were treated with either rabbit IgG or anti-CX3CR1 at 50 µg/ml prior to sequential activation. Data were reproduced in three experiments. d Analysis of the effect of CX3CR1 blockade on transmigration of monocyte subpopulations through HUVECs activated by TNF or TNF + VEGF. n = 3 experiments, data are mean ± s.d., two-way ANOVA with Holm-Sidak adjustment. e, f Analysis of the influence of CX3CR1 blockade on extravasation of adoptively transferred human monocytes to mouse peritoneum in thioglycolate +Tnf− (e) and thioglycolate +TnfVegf-a- (f) induced peritonitis. Peritonitis was induced as described in the schematic in Supplementary Fig. 11. Isolated monocytes were treated with either IgG or anti-CX3CR1 antibody at 50 µg/ml (with/without PTX) prior to mouse injection, n = 6 mice per group pooled from two experiments, mean ± s.d., ANOVA. g Effect of CX3CR1 blockade on HPMo recruitment to SKBR7 tumour xenografts. Human monocyte recruitment to the xenografts was performed as described in Fig.1 with human monocytes treated with the antibodies at 50 µg/ml (with/without PTX) before mouse injection. Left: Percentage of HPMo within recruited human monocytes, n = 9 mice per group injected with monocytes from 3 donors. Data are mean ± s.d., *p-value <0.05, paired t-test. Right: quantification of the percentage of total human monocytes within tumour cell suspension, n = 9 mice per group injected with monocytes from 3 donors. Data are mean ± s.d., *p-value <0.05, ANOVA
