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.

The subset of human blood monocytes expressing low levels of CD14 and high levels of CD16 (CD14 ؉ CD16 ؉ ) exhibits features resembling mature tissue macrophages and can be expanded in inflammatory conditions. We analyzed expression of CC chemokine receptors (CCR) in CD14 ؉ CD16 ؉ versus CD14 ؉؉ monocytes, which may be crucial for specific trafficking. Multicolor flow cytometric analysis of whole peripheral blood revealed that, as opposed to CD14 ؉؉ monocytes, the CD14 ؉ CD16 ؉ subset lacked surface expression of monocyte chemotactic protein-1 (MCP-1) receptor CCR2, however, it showed significantly higher surface expression of the macrophage inflammatory protein 1␣ (MIP-1␣)/RANTES receptor CCR5. This was paralleled by differences in mRNA expression in the subsets, as shown by reverse transcriptase-polymerase chain reaction using sorted cells. In comparison to CD14 ؉؉ monocytes, CD14 ؉ CD16 ؉ cells expressed lower CCR2 but higher CCR5 transcript levels, whereas CCR1 levels were equivalent. Flow cytometric analysis of isolated human monocytes recovered after transendothelial chemotaxis assays revealed that the percentage of CD14 ؉ CD16 ؉ cells was dramatically reduced in the fraction migrating toward MCP-1 compared with the fraction that did not migrate or the input, showing that polarized CCR2 expression was accompanied by a differential chemotactic responsiveness. Moreover, CD11b surface expression was preferentially up-regulated by MCP-1 in CD14 ؉؉ cells but by MIP-1␣ in CD14 ؉ CD16 ؉ monocytes, confirming the functional relevance of distinct CCR expression. The characteristics of CD14 ؉ CD16 ؉ cells may reflect preactivation by cytokines and determine their predilective localization during specific inflammatory conditions or susceptibility to infection. J. Leukoc. Biol. 67: 699-704; 2000.

Healthy donors infused with high doses of glucocorticoids [GCs; methyl-prednisolone (MP); 500 mg/day for 3 days] suffer a selective depletion of the CD14(+)CD16(+) monocytes such that these cells are reduced by 95% on day 5. In vitro studies revealed that at 11 h of culture in the presence of 10(-)(5) M MP, no depletion was observed as yet, but a reduction by 80% was seen after 24 h. In dose-response analysis, MP still led to a 50% reduction of CD14(+)CD16(+) monocytes at 10(-)(7) M. Depletion could not be overcome by addition of the cytokines interleukin-1beta or macrophage-colony stimulating factor, and it was independent of CD95. Depletion was, however, inhibited by the caspase 3,8 blocker z-Val-Ala-Asp, suggesting that cell death occurs in a caspase-dependent manner. Furthermore, blockade of depletion by RU-486 indicates that the intracellular GC receptor (GCR) is involved. Measurement of GCR by flow cytometry revealed a 50% higher level of expression in the CD14(+)CD16(+) monocytes. Our studies show a selective depletion of CD14(+)CD16(+) monocytes by GC treatment in vivo and in vitro, an effect to which the modestly increased level of GCR may contribute.

In human peripheral blood the classical CD14++DR+ monocytes and the pro‐inflammatory CD14+CD16+DR++ monocytes can be distinguished. In erysipelas we found strongly increased numbers of CD14+CD16+ monocytes on the day of diagnosis (day 1) in 11 patients with an average of 150.5±76.0 cells/μl, while 1 patient had low levels (35 cells/μl, control donors 48.8±19.8 cells/μl). The classical monocytes were only moderately elevated in the erysipelas patients (factor 1.7 as compared to controls). Patients exhibited increased body temperature, erythrocyte sedimentation rate and increased serum levels for C‐reactive protein (CRP), IL‐6 and macrophage‐colony‐stimulating factor. Among these, body temperature and CRP showed a significant correlation to the numbers of CD14+CD16+ monocytes. In 4 of 4 patients with high levels of CD14+CD16+ monocytes, these levels returned to that seen in controls by day 5 of antibiotic therapy. Determination of intracellular TNF was performed by three‐color immunofluorescence and flow cytometry after ex vivo stimulation withlipoteichoic acid, a typical constituent of streptococci. Here, patient CD14+DR++ pro‐inflammatory monocytes showed a twofold lower level of intracellular TNF. By contrast, expression of TNF was unaltered in the classical CD14++ monocytes. These data show that in erysipelas the pro‐inflammatory CD14+CD16+DR++ monocytes are substantially expanded and selectively tolerant to stimulation by streptococcal products.

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