The Proinflammatory CD14+CD16+DR++ Monocytes Are a Major Source of TNF

TLDR

Human blood contains two monocyte subsets—classical CD14++CD16−DR+ cells and a 10 % proinflammatory CD14+CD16+DR++ population. We quantified TNF production in these subsets by three‑color immunofluorescence and flow cytometry on whole peripheral blood stimulated with LPS or the TLR2 ligand Pam3Cys. Stimulation with LPS induced a three‑fold higher TNF signal in proinflammatory monocytes, while Pam3Cys triggered a ten‑fold increase; TLR2 expression was twice as high on these cells, and depletion of CD16+ monocytes reduced LPS‑ and Pam3Cys‑induced TNF by 28 % and 64 %, confirming that the minor CD14+CD16+DR++ subset is the major TNF producer in human blood.

Abstract

In human blood two monocyte populations can be distinguished, i.e., the CD14(++)CD16(-)DR(+) classical monocytes and the CD14(+)CD16(+)DR(++) proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14(+)CD16(+)DR(++) monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16(+) monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14(+)CD16(+) monocytes are major producers of TNF in human blood.