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Institute for Immunology, University of Muenchen, and
GSF National Research Center for Environment and Health, Institute of Molecular Immunology, Muenchen, Germany;
Clinical Cooperation Group, Aerosols in Medicine, Institute for Inhalationbiology, GSF National Research Center for Environment and Health and Asklepios-Fachkliniken Muenchen-Gauting, Gauting, Germany;
Institute of Cancer Research and Molecular Biology, Medisinsk Teknisk Senter, Trondheim, Norway; and
¶ Department for Microbiology and Immunology, University of Leicester, Leicester, United Kingdom
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)-Lys4-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.
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