Coexpression of tissue factor and tissue factor pathway inhibitor by human monocytes 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 fact that this technique has been used for many years to isolate monocytes for cellular immunotherapy, it is not known whether the process of elutriation can stimulate tissue factor (TF) expression and therefore trigger coagulation 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.


METHODS
Human monocytes are separated by leukapheresis and elutriation in sterile, endotoxin-free conditions. TF and TFPI mRNA is detected by reverse transcription-polymerase chain reaction. TF and TFPI are measured by enzyme-linked immunosorbent assay in cell lysates. TF antigen expression on cell surface is evidenced by direct-flow cytometry. Two functional tests (a chronometric test and an amidolytic assay) assess the capacity of monocytes to trigger thrombin generation. The response to lipopolysaccharide (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 expression of TF mRNA is dramatically increased by LPS activation. This is correlated with a 100-fold increase in the amount of TF antigen in monocyte lysates. Flow immunocytometry confirms the expression of TF antigen on cell membrane 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 potential to trigger thrombin generation in response to LPS.


CONCLUSIONS
In spite of the coexpression of TF mRNA and TFPI mRNA, elutriated monocytes are capable of supporting prothrombinase activity. This should be taken into account in the evaluation of the safety of adoptive cellular immunotherapy.
Nguyên
P
P
Laboratoire d'Hématologie, Institut Federatif de Rescherche 53 Bimolécules, Université de Reims Champagne-Ardenne, France. pnguyen@chu-reims.fr
Broussas
M
M
Cornillet-Lefèbvre
P
P
Potron
G
G
D000941:Antigens / Q000097:blood; D002448:Cell Adhesion / Q000187:drug effects; D016238:Cytapheresis / Q000379:methods*; D006490:Hemostatics / Q000378:metabolism; D006801:Humans; D007937:Leukapheresis; D008070:Lipopolysaccharides / Q000494:pharmacology; D008074:Lipoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013917:Thrombin / Q000096:biosynthesis; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000941:Antigens; D006490:Hemostatics; D008070:Lipopolysaccharides; D008074:Lipoproteins; D012333:RNA, Messenger; C051928:lipoprotein-associated coagulation inhibitor; D013925:Thromboplastin; D013917:Thrombin