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. 2017 Jun 26;7:4230. doi: 10.1038/s41598-017-04275-7

Figure 5.

Figure 5

Role of CD32a and mCD14 in LTF-IC-mediated monocyte activation. Human monocytes were stimulated with 30 μg/ml huLTF-M860-IC (A), or RA-IgG-IC (B), in the presence or absence (Med) of blocking mAbs (15 μg/ml) against CD16 (B73.1, eBioscience), or CD32 (IV.3, eBioscience), or CD64 (10.1, Stem Cell Tech) for 18 h, followed by quantitation of TNF-α in the culture supernatant by ELISA. (C) Monocytes stimulated with 3 μg/ml LPS in the presence or absence (Med) of anti-CD32a mAb (15 μg/ml), or isotype control Abs as controls. (D) Human monocytes were stained with 30 μg/ml M860-IC containing FITC-conjugated huLTF in the presence or absence (M860-IC) of 10 μg/ml soluble recombinant human CD14 (M860-IC + sCD14) or OVA (M860-IC + OVA) for 1 h at 4 °C, followed by FACS analysis. Unstained cells were included as negative control (filled histogram). (E) Human monocytes were treated with 30 μg/ml huLTF-M860 IC in the presence of mouse mAbs (15 μg/ml) against human LRP-1 (αLRP1), nucleolin-1 (αNucleolin), or CD14 (αCD14) for 18 h, followed by quantitation of TNF-α levels in the culture supernatants by ELISA. Cells treated with 1 μg/ml PMA in the presence of αCD14 mAb or isotype control mAb were included for specificity controls. (F) The anti-CD14 mAb was titrated against LTF-M860 IC (30 μg/ml) in monocyte activation assays with mouse IgG1 (1 μg/ml) as isotype control. Readings from unstimulated cells (Med) are also indicated in the figure. (G) TLR4-specific inhibitor CLI-095 was titrated against LTF-M860 IC (30 μg/ml) in monocyte activation assays monitoring TNF-α production. Monocytes stimulated with 5 μg/ml zymosan in the presence or absence of 1 μg/ml CLI-095 were included as specificity control. For ELISAs, values are mean TNF-α concentration (ng/ml) ± SEM from triplicate cultures. *p < 0.05, **p < 0.01 compared with M860-IC- or LPS-stimulated cells in the presence of isotype control Abs or in the absence of inhibiting agents. NS: not statistically significant. Results are representative of three independent experiments.