Separation of dendritic cells from highly purified human monocytes by counterflow centrifugation induces tissue factor expression

BACKGROUND: In vitro generation of dendritic cells (DCs) from human monocyt es represents a promising tool in immunotherapy. However, it is not known w hether the separation of DCs from monocytes induces tissue factor expressio n and therefore may trigger coagulation in patients receiving these DC prep arations. The aim of this study is thus to analyze tissue factor expression on monocyte-derived DCs and to compare their ability to trigger thrombin g eneration to that of macrophages obtained from the same monocytes. STUDY DESIGN AND METHODS: Human monocytes are separated by leukapheresis an d washed by using counterflow centrifugation in sterile, endotoxin-free con ditions. Macrophages are grown from human monocytes in the presence of GM-C SF alone and immature DCs are grown in the presence of GM-CSF plus IL-4 for 5 days with fetal calf serum (IDC-FCS). Immature DCs are also grown from h uman monocytes for 7 days in the presence of GM-CSF plus IL-4 with human gr oup AB serum (IDC-HS). The addition of prostaglandin E-2 and TNF alpha in t his culture medium at Day 5 leads to mature DCs (MDC-HS). Tissue factor mRN A expression is studied by RT-PCR analysis. Tissue factor antigen is measur ed by ELISA in cell lysates and by direct flow cytometry. The procoagulant activity of intact cells is assessed by using an amidolytic assay or a chro nometric assay. RESULTS: IDC-FCS express tissue factor mRNA and antigen and trigger thrombi n generation. Procoagulant activity of IDC-FCS is dependent on both tissue factor expression and exposure to anionic phospholipid. Monocyte-derived ma crophages cultured for 5 days with GM-CSF alone express lower levels of tis sue factor mRNA, tissue factor antigen, and procoagulant activity than IDC- FCS. IDC-HS and MDC-HS also express high levels of tissue factor mRNA and a ntigen and support procoagulant activity. CONCLUSION: Monocyte-derived DCs express a high level of functional tissue factor and support procoagulant activity. This finding should be taken into account in clinical trials.