The influence of different anticoagulants and sample preparation methods on measurement of mCD14 on bovine monocytes and polymorphonuclear neutrophil leukocytes

<p>Abstract</p> <p>Background</p> <p>Membrane-CD14 (mCD14) is expressed on the surface of monocytes, macrophages and polymorphonuclear neutrophil leukocytes (PMN). mCD14 acts as a co-receptor along with Toll like receptor 4 (TLR 4) and MD-2 for the detection of lipopolysaccharide (LPS). However, studies using different sample preparation methods and anticoagulants have reported different levels of mCD14 on the surface of monocytes and neutrophils. In this study, the influence of various anticoagulants and processing methods on measurement of mCD14 on monocytes and neutrophils was examined.</p> <p>Results</p> <p>Whole blood samples were collected in vacutainer tubes containing either sodium heparin (HEPARIN), ethylenediaminetetraacetic acid (EDTA) or sodium citrate (CITRATE). mCD14 on neutrophils and monocytes in whole blood samples or isolated cells was measured by the method of flow cytometry using fluorescein isothiocyanate (FITC)-labeled monoclonal antibody. There was a significant difference (<it>p </it>< 0.05) in the mean channel fluorescence intensity (MFI) of mCD14 on neutrophils in whole blood samples anticoagulated with HEPARIN (MFI = 64.77) in comparison with those in whole blood samples anticoagulated with either EDTA (MFI = 38.25) or CITRATE (MFI = 43.7). The MFI of mCD14 on monocytes in whole blood samples anticoagulted with HEPARIN (MFI = 206.90) was significantly higher than the MFI in whole blood samples anticoagulated with EDTA (MFI = 149.37) but similar to that with CITRATE (MFI = 162.55). There was no significant difference in the percentage of whole blood neutrophils or monocytes expressing mCD14 irrespective of type of anticoagulant used. However, MFI of mCD14 on monocytes was about 3.2-folds (HEPARIN), 3.9-folds (EDTA) or 3.7 folds (CITRATE) higher than those on neutrophils. Furthermore, there was no significant difference in mCD14 levels between unprocessed whole blood monocytes and monocytes in peripheral blood mononuclear cell preparation. Conversely, a highly significant difference was observed in mCD14 between unprocessed whole blood neutrophils and isolated neutrophils (<it>p </it>< 0.05).</p> <p>Conclusion</p> <p>From these results, it is suggested that sodium heparin should be the preferred anticoagulant for use in the reliable quantification of the surface expression of mCD14. Furthermore, measurement of mCD14 is best carried out in whole blood samples, both for neutrophils and monocytes.</p

Surface phenotype analysis of CD16+ monocytes from leukapheresis collections for peripheral blood progenitors

In peripheral blood progenitor cell (PBPC) collections from patients with solid tumour or haematological malignancy, monocytes were separated into two subpopulations. The majority of monocytes expressed CD14 at a high density without CD16 antigen (the CD14+CD16− monocytes). The remaining monocytes co-expressed CD14 and CD16 (the CD14+CD16+ monocytes). These CD14+CD16+ monocytes amounted to 20.6 ± 15.8%, while those in peripheral blood (PB) obtained from healthy volunteers were 7.3 ± 3.1% (P < 0.05). When subdividing the CD14+CD16+ monocytes into CD14brightCD16dim and CD14dimCD16bright cells, both populations were found to be increased in PBPC collections. Since typical CD14+CD16+ monocytes are the CD14dimCD16bright population, we compared the additional surface antigens on CD14dimCD16bright monocytes with those of CD14+CD16−monocytes. In PBPC collections, the CD14dimCD16bright monocytes exhibited lower levels of CD11b, CD15, CD33 and CD38 expression and higher levels of CD4, CD11a, CD11c and MHC class II, and also revealed a higher percentage of CD4+ cells and a lower percentage of CD15+ cells and CD38+ cells, compared with the CD14+CD16− monocytes. When compared with the CD14dimCD16bright monocytes in PB, those in PBPC collections exhibited higher expression of CD4 and lower expression of CD11b, and also showed higher percentages of CD4+ cells and CD38+ cells and a lower percentage of CD11b+ cells. These results suggest that PBPC collections may be rich in the CD14+CD16+ monocytes in which the proportion of the immature population is increased. It is likely that these monocytes participate in the haematological and immune recovery after PBPC transplantation

Selective depletion of CD14+ CD16+ monocytes by glucocorticoid therapy

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on many cells of the body, including monocytes. Here we show that a 5-day course of high dose GC therapy differentially affected the CD14++ and the CD14+ CD16+ monocyte subpopulations in 10 patients treated for multiple sclerosis. While the classical (CD14++) monocytes exhibited a substantial increase from 495 ± 132 to 755 ± 337 cells/μl, the CD14+ CD16+ monocytes responded with a pronounced decrease from 36 ± 15 to 2 ± 3 cells/μl (P < 0.001). In 4/10 patients the CD14+ CD16+ monocytes fell below detection limits (< 0.2 cells/μl). This observation was confirmed when the CD14+ CD16+ monocytes were identified by virtue of their low CD33 expression as these cells decreased as well. After discontinuation of GC therapy the CD14+ CD16+ monocytes reappeared and reached normal levels after 1 week. The profound depletion of CD14+ CD16+ monocytes by GC as described here is a novel effect of GC action in vivo and may contribute to GC-mediated immunosuppression. Determination of the number of this monocyte subset may also serve to monitor the effectiveness of GC therapy in patients requiring immunosuppressive treatment

Tissue-specific induction of ADAMTS2 in monocytes and macrophages by glucocorticoids.

The regulated expression of ADAMTS2 (a disintegrin and metalloproteinase with thrombospondin motifs), a secreted metalloproteinase involved in the processing of procollagen to collagen, was studied in peripheral blood mononuclear cells (PBMC). Stimulation with glucocorticoids (GC) resulted in a pronounced dose- and time-dependent increase of ADAMTS2 mRNA levels in PBMC. The increase of ADAMTS2 expression was specific for CD14++ monocytes (440-fold) and alveolar macrophages (200-fold), whereas CD3+ (T lymphocytes), phytohemagglutinin-activated CD3+ (T lymphocytes), and CD19+ (B lymphocytes) showed no significant changes in ADAMTS2 mRNA after GC treatment. Treatment of monocyte-derived macrophages (MDM) with GC also resulted in an increase of ADAMTS2 protein in the culture tissue media. Using the GC analog RU486, GC-mediated induction of ADAMTS2 mRNA was blocked, implicating that GC acts specifically via the GC-receptor. In agreement with findings in blood monocytes, cell lines of the monocytic lineage (MM6, THP-1) showed significant GC-induced significant increases in ADAMTS2 mRNA, while in epithelial cells (A549, Calu-3, Colo320, BT-20) and fibroblast (MRC-5, WI-38, and two NHDF-c cell types from adult cheek and upper arm), they showed no or little responsiveness to GC. As macrophages have important functions in immune defense and tissue homeostasis, these findings suggest that GC-mediated specific induction of ADAMTS2 in these cells may play a crucial role in the resolution of inflammation and wound repair

Regulation of the expression of Fcγ receptor on circulating neutrophils and monocytes in Kawasaki disease

To investigate the regulation of Fcγ receptor (FcγR) expression on circulating phagocytes in Kawasaki disease (KD), we analysed the expressions of FcγRI, II and III on neutrophils and monocytes in 20 patients with KD, 10 with a bacterial infection (BI), 10 with a viral infection (VI), and 10 healthy controls (HC) using flow cytometric analysis. The KD patients had a significantly higher level of FcγRI expression on neutrophils, but not on monocytes, than the BI, VI and HC patients. FcγRII expression on neutrophils was significantly higher in KD, BI and VI than HC, but there was no significant difference in FcγRII expression among KD, BI and VI. FcγRIII expression on neutrophils in KD was significantly lower than in VI and HC, but was higher on monocytes. A kinetic analysis of FcγR expression in KD demonstrated the expression of FcγRI and II on neutrophils to decline, but no remarkable change was observed in the monocytes, from the subacute phase through the convalescent phase. In addition, FcγRIII expression on neutrophils increased, while FcγRIII expression on monocytes decreased during the time course of KD. FcγR expression in the acute phase of KD is thus characterized by markedly increased expression of FcγRI on neutrophils, followed by a subsequent decrease, and decreased expression of FcγRIII on neutrophils and increased expression of FcγRIII on monocytes followed by a reverse kinetics during the clinical course. These findings are thus considered to reflect the functional up-regulation of neutrophils and monocytes in KD