TNF-α Autocrine Feedback Loops in Human Monocytes: The Pro- and Anti-Inflammatory Roles of the TNF-α Receptors Support the Concept of Selective TNFR1 Blockade In Vivo

Figure 5

An example of flow cytometry overlay histograms. Overlay histograms are shown for gated monocytes from one subject. (a) Four histograms are shown, with fluorescence intensity in the PE channel on the -axis and cell count on the -axis. The black histogram represents LPS-stimulated monocytes incubated with a PE-labelled isotype control antibody. The red histogram represents monocytes stimulated with LPS and labelled with PE-TNFR1 mAb. Green and pink histograms represent LPS-stimulated monocytes incubated in the presence of TNF-α mAb or TNFR2 mAb, respectively, and then labelled with PE-TNFR1 mAb. The altered positions of the histograms illustrate the effect of TNF-α or TNFR2 blockade on subsequent TNFR1 expression. (b) Four histograms are shown, with fluorescence intensity in the FITC channel on the -axis and cell count on the -axis. The black histogram represents LPS-stimulated monocytes incubated with a FITC-labelled isotype control antibody. The red histogram represents monocytes stimulated with LPS and labelled with FITC-TNFR2 mAb. Green and pink histograms represent LPS-stimulated monocytes incubated in the presence of TNF-α mAb or TNFR1 mAb, respectively, and then labelled with FITC-TNFR2 mAb. Red, green, and pink histograms overlay each other exactly illustrating that in this subject’s monocytes there was no effect of TNF-α or TNFR1 blockade on subsequent TNFR2 expression. Each of the three experimental conditions was conducted alongside samples subjected to the same conditions but incubated with an isotype control flow cytometry antibody. As all isotype control histograms overlaid each other, only one of three is shown in each graph for ease of visualisation.

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