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The CD14+/CD16+ cells account for about 10% of all blood monocytes. They are characterized by a low level expression of the CD14 molecule and a high level expression of the CD16 (Fc gamma R III) molecule. Polymerase chain reaction analysis of mRNA prevalence in CD14+/CD16+ cells (compared to the regular CD14++ blood monocytes) demonstrates low levels of CD14 transcripts and high levels of CD16 transcripts, suggestive of a transcriptional control for both of these proteins. Analysis of additional cell surface molecules in three-color immunofluorescence reveals that CD14+/CD16+ cells express the Fc gamma R II in all, and Fc gamma R I and ICAM-1 in some donors. Furthermore, class II antigens are expressed at fourfold higher levels, while both, CD11b and CD33 cell surface proteins, are decreased by a factor of two. Transcript levels were reduced in CD14+/CD16+ cells for all three cell surface molecules. Since these phenotypic markers of the CD14+/CD16+ blood monocytes are reminiscent of tissue macrophages, we performed a comparative analysis with alveolar macrophages (AM). These cells are similar to the CD14+/CD16+ monocytes in that they show low levels of CD14 and strong expression of CD16. Furthermore, similar to the CD14+/CD16+ cells, the AM also exhibit higher levels of class II and lower levels of CD11b and CD33 when compared to the regular CD14++ blood monocytes. In vitro induction of maturation of blood monocytes by 5 day culture of peripheral blood mononuclear cells in 10% human serum will result in decreased CD14 and increased CD16 cell surface expression on the monocyte derived macrophages. At the same time, these cells acquire increased levels of class II and decreased levels of CD11b and CD33. Taken together, these data show that CD14+/CD16+ monocytes, while still in circulation, have acquired features in common with mature tissue macrophages.

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.

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Cell adhesion to endothelial cells stimulated by tumor necrosis factor-a (TNF) is due to induction of surface receptors, such as vascular cell adhesion molecule-1 (VCAM-1). The antiaxidant pyrrolidine dithiocarbamate (PDTC) specifically inhibits activation of nuclear factor-KB (NF-KB). Since *B motifs are present in VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) promoters, we used PDTC to study the regulatory mechanisms of VCAM-1 and ICAM-1 induction and subsequent monocyte adhesion in TNF-treated human umbilical vein endothelial cells (HUVECs). PDTC or JV-acetylcysteine dose dependently reduced TNF-induced VCAM-1 but not ICAM-1 surface protein (also in human umbilical arterial endothelial cells) and mRNA expression (by 70% at 100 junol/L PDTC) in HUVECs as assessed by flow cytometry and porymerase chain reaction. Gel-shift analysis in HUVECs demonstrated that PDTC prevented NF-KB mobilization by TNF, suggesting that only VCAM-1 induction was controlled by NF-KB. Since HUVECs released superoxide anions in response to TNF, and H 2 O 2

IL-10 is a unique cytokine because it is anti-inflammatory and immunosuppressive. IL-10 is regulated at the level of transcription, but the critical motifs and the relevant transcription factors controlling this gene have remained elusive to date. We now report that a sequence at −120 bp in the human IL-10 promoter binds Stat3 but no other Stat proteins. Mutation of this motif abrogates LPS-induced trans-activation. Overexpression of dominant negative Stat3 suppresses promoter activity, while wild-type Stat3 leads to an enhancement of this activity. Our results show that Stat3, by binding to a single motif in the IL-10 promoter, is controlling expression of the human IL-10 gene.

Strenuous, anaerobic exercise leads to an increase of leukocytes that are mobilized from the marginal pool. We have analyzed in human peripheral blood the effect of exercise on the number of CD14(+)CD16(+) monocytes as determined by two-color immunofluorescence and flow cytometry. We show herein that this type of monocyte responds with a dramatic up to 4.8-fold increase. Mobilization does not occur after 1 min at 100 or 200 W but 1 min at 400 W leads to a twofold increase of the CD14(+)CD16(+) monocytes immediately after exercise. The numbers remain high at 5 min and gradually decrease to reach the initial level at 20 min postexercise. After 20 min of rest, the CD14(+)CD16(+) monocytes can be mobilized again by a second exercise. The CD14(+)CD16(+) monocytes appear to be mobilized from the marginal pool where they preferentially home because of a higher expression of adhesion molecules like CD11d and very late antigen-4. Exercise goes along with an increase of catecholamines, and mobilization of the CD14(+)CD16(+) monocytes can be substantially reduced by treatment of donors with the beta-adrenergic receptor blocker propranolol. Mobilization of CD14(+)CD16(+) monocytes by a catecholamine-dependent mechanism may contribute to the increase of these cells in various clinical conditions.