Coexpression of tissue factor and tissue factor pathway inhibitor by humanmonocytes purified by leukapheresis and elutriation. Response of nonadherent cells to lipopolysaccharide

BACKGROUND: Counterflow centrifugal elutriation is the method of choice for obtaining a large quantity of highly purified monocytes. In spite of the f act that this technique has been used for many years to isolate monocytes f or cellular immunotherapy, it is not known whether the process of elutriati on can stimulate tissue factor (TF) expression and therefore trigger coagul ation in patients receiving these cell preparations. The aim of the present study is thus to identify TF and TF pathway inhibitor (TFPI) in elutriated monocytes and to evaluate their ability to trigger thrombin generation. STUDY DESIGN AND METHODS: Human monocytes are separated by leukapheresis an d elutriation in sterile, endotoxin-free conditions. TF and TFPI mRNA is de tected by reverse transcription-polymerase chain reaction. TF and TFPI are measured by enzyme-linked immunosorbent assay in cell lysates. TF antigen e xpression on cell surface is evidenced by direct-flow cytometry. Two functi onal tests (a chronometric test and an amidolytic assay) assess the capacit y of monocytes to trigger thrombin generation. The response to lipopolysacc haride (LPS) is evaluated with each technique. Monocytic cell line THP-1 is used as a positive control. RESULTS: Elutriated monocytes coexpress TF mRNA and TFPI mRNA. The expressi on of TF mRNA is dramatically increased by LPS activation. This is correlat ed with a 100-fold increase in the amount of TF antigen in monocyte lysates . Flow immunocytometry confirms the expression of TF antigen on cell membra ne in response to LPS stimulation, whereas TFPI mRNA is slightly increased after LPS stimulation. The amount of TFPI antigen in cell lysates is small when compared to that in plasma. Elutriated monocytes have a strong potenti al to trigger thrombin generation in response to LPS. CONCLUSION: In spite of the coexpression of TF mRNA and TFPI mRNA, elutriat ed monocytes are capable of supporting prothrombinase activity. This should be taken into account in the evaluation of the safety of adoptive cellular immunotherapy.