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 4

Autocrine effects of TNF-α on TNF-α receptor cell surface expression and soluble receptor concentration. In (a) and (b), the surface expression of TNFR1 and TNFR2 on monocytes stimulated with LPS (100 ng/mL) ± TNF-α mAb or selective TNF-α receptor blockade (25 μg/mL) for 20 hours was determined (e.g., TNFR1 expression was determined in monocytes where TNF-α signalling via TNFR2 had been prevented with a blocking mAb). Concentrations were compared to LPS-stimulated cells, using a Wilcoxon Signed-Ranks Test. In (c) and (d), time course profiles of TNFR1 MFI and TNFR2 MFI, respectively, are shown in LPS-stimulated monocytes expressing one or both types of receptor. No overall difference was observed for time course profiles of TNFR1 expression in cells expressing solely TNFR1 (Friedman’s ) nor cells expressing both receptors (Friedman’s ). Friedman’s test was significant for TNFR2 expression in cells expressing TNFR2 solely () and cells expressing both receptors (). Significant post hoc pairwise comparisons values are shown in the figures. Differences in MFI at each time point between cell types were assessed with a Wilcoxon Signed-Ranks Test. In (e) TNFR1 and (f) TNFR2, the columns show the concentration in the supernatant of LPS-stimulated monocytes (for 22 hours) ± TNF-α mAb (10 μg/mL). Concentrations were compared to the positive control, LPS-stimulated cells, using a Wilcoxon Signed-Ranks Test. Columns in all figures show median (IQR) values. The experiments represent data from 6 subjects. Experimental conditions/time points for flow cytometry analyses were conducted in duplicate (a–d) with the exception of freshly isolated monocytes (time: 0 hours) in (c) and (d) due to limitations to cell numbers available. Experimental conditions in (e) and (f) were conducted in culture wells in duplicate and in each sample run in duplicate on the subsequent ELISA.

(a)

(b)

(c)

(d)

(e)

(f)