Arachidonic Acid Enhances the Tissue Factor Expression of Mononuclear Cells by the Cyclo-Oxygenase-1 Pathway: Beneficial Effect of n-3 Fatty Acids This information is current as of September 27, 2021. Yves Cadroy, Dominique Dupouy and Bernard Boneu J Immunol 1998; 160:6145-6150; ; http://www.jimmunol.org/content/160/12/6145 Downloaded from References This article cites 32 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/160/12/6145.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Arachidonic Acid Enhances the Tissue Factor Expression of Mononuclear Cells by the Cyclo-Oxygenase-1 Pathway: Beneficial Effect of n-3 Fatty Acids1 Yves Cadroy,2 Dominique Dupouy, and Bernard Boneu Monocytes express tissue factor (TF) upon stimulation by inflammatory agents. Dietary administration of fish oil rich in eicosa- pentaenoic acid (EPA) and docosahexaenoic acid (DHA) results in an impairment of TF expression by monocytes. EPA and DHA are metabolized differently from arachidonic acid (AA), the major fatty acid present in cell membranes. We examined the effects of AA on the TF expression of isolated human PBMC, and we determined whether EPA and DHA modulated this phenomenon differently. Nonstimulated PBMC had a low TF-dependent procoagulant activity. When PBMC were incubated with increasing concentrations of AA, the TF-dependent procoagulant activity increased in a dose-dependent manner to 190% at 7.5 mM. Indo- Downloaded from methacin, a cyclo-oxygenase inhibitor, totally abolished the stimulating effect of AA, whereas specific pharmacologic inhibitors of cyclo-oxygenase-2 or of 5-lipoxygenase had no inhibitory effect. A thromboxane (TX)A2/endoperoxides receptor antagonist and a TX synthase inhibitor blocked the potentiating effect of AA. Purified PGG2 and carbocyclic TXA2, a TXA2 agonist, enhanced the procoagulant activity of PBMC in a dose-dependent manner whereas, in contrast, PGE2 inhibited it. Finally, contrary to AA, EPA or DHA did not increase TXB2 production or TF expression by PBMC. The TF-dependent procoagulant activity of isolated PBMC http://www.jimmunol.org/ was increased by AA through the production of cyclo-oxygenase-1 metabolites; the combined action of PGG2 and TXA2, which potentiated it, was greater than that of PGE2, which inhibited it. Dietary n-3 fatty acids exert part of their beneficial effect by modulating this procoagulant activity differently from AA. The Journal of Immunology, 1998, 160: 6145–6150. rachidonic acid (AA;3 20:4, n-6) is a polyunsaturated atherosclerosis. The molecular mechanisms by which dietary n-3 fatty acid present in cell membranes that modulates di- fatty acids exert their beneficial effects are complex and have not A verse physiologic and pathologic responses. These ef- been totally clarified. Some of these effects have been attributed to fects are primarily mediated by its metabolic products, eico- the activity of n-3 fatty acids on monocytes (4, 8, 11–13). sanoids. Eicosanoids include notably the cyclo-oxygenase Monocytes are centrally involved in numerous pathophysiologic by guest on September 27, 2021 products, i.e., PG and TX, and the lipoxygenase products, i.e., processes, such as thrombosis, atherosclerosis, wound repair, and leukotrienes and hydroxyeicosatetraenoic acids (HETEs). Their ef- inflammation. These properties are partly related to their ability to fects, which depend on the nature of compounds, principally con- express various procoagulant activities (14–17). The procoagulant cern inflammation and thromboregulation (1). Eicosapentaenoic activities of monocytes are mediated to a large extent by cell sur- acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3) face-associated tissue factor (TF). TF is the cellular receptor and are other polyunsaturated fatty acids contained in fish oil. EPA is cofactor for plasma factor VII(a), which initiates the coagulation metabolized into eicosanoids that are different from the corre- protease cascade leading ultimately to the generation of thrombin sponding compounds derived from AA and are considered to de- and fibrin (18). Dietary administration of fish oil results in an im- velop reduced biologic activities (2–5). DHA is not metabolized by pairment of TF expression by monocytes (8, 12, 13). The molec- cyclo-oxygenase or lipoxygenase enzymes (6). Experimental (7, ular mechanisms underlying TF activity reduction after n-3 fatty 8), epidemiologic, and interventional (9, 10) studies suggest that acids intake are unknown. However, since monocytes are impor- consumption of fish oil, which causes a progressive decrease in the tant generators of eicosanoids and possess both cyclo-oxygenase AA content of tissue phospholipids and an increase in the tissue and lipoxygenase enzymes, changes in the monocyte capacity to level of n-3 fatty acids, exerts beneficial effects on thrombosis and generate eicosanoids by these pathways have been suspected to influence monocyte TF activity. Thus, several recent studies have indicated that eicosanoids may modulate the procoagulant proper- Laboratoire de Recherche sur l’He´mostase et la Thrombose, Centre Hospitalo-Uni- ties of monocytes. For example, prostacyclin analogues and PGE1 versitaire Purpan, Toulouse, France cause a decrease in cytokine-induced TF activity by monocytes Received for publication October 3, 1997. Accepted for publication February (19, 20), whereas, conversely, platelet 12-HETE enhances PBMC 12, 1998. procoagulant activity (21). The costs of publication of this article were defrayed in part by the payment of page To clarify the mechanisms of the beneficial effect of dietary fish charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. oil on the procoagulant activity of monocytes, we investigated in 1 This work was supported in part by a grant from Pierre Fabre, Castres, France. vitro the effects of AA, EPA, and DHA on TF expression by iso- 2 Address correspondence and reprint requests to Dr. Yves Cadroy, Laboratoire lated PBMC. We show for the first time that AA, but not EPA or d’He´matologie, Pavillon Lefe`bvre, Centre Hospitalo-Universitaire Purpan, 31059 DHA, enhanced the TF-dependent procoagulant activity of PBMC. Toulouse Cedex, France. The roles of the cyclo-oxygenase and lipoxygenase pathways were 3 Abbreviations used in this paper: AA, arachidonic acid; TX, thromboxane; HETE, also examined. We describe a novel role for the cyclo-oxygenase-1 hydroxyeicosatetraenoic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic pathway in modulating the TF expression of AA-stimulated acid; TF, tissue factor; CTXA2, carbocyclic thromboxane A2; AU, procoagulant ac- tivity units. PBMC that appeared to be balanced by the opposite effects of Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 6146 ARACHIDONIC ACID AND MONONUCLEAR CELL TISSUE FACTOR PGE2, which inhibited it, and endoperoxides/TXA2, which Determination of TF, TXB2, and PGE2 potentiated it. TF Ag was measured on cell lysates by commercially available ELISA (Imubind Tissue Factor, American Diagnostica). Cell lysates were pre- pared by lysing PBMC in PBS containing 1% Triton X-100, 1 mM EDTA Materials and Methods (Merck, Chelles, France), 16 mM octyl PD glucopyranoside (Boehringer Cell isolation and cell cultures Mannheim, Meylan, France), 10 mM pepstatin A, 10 mM leupeptin, 0.1 mM PMSF (Sigma), and 100 kallkrein inhibitor units/ml aprotinin (Sanofi- PBMC were isolated using a modification of the method described by Choay, Paris, France). The cell lysates were then frozen and thawed three 2 Balter et al. to efficiently deplete platelet from the cell preparation (22). times. They were stored at 80°C until assayed. TXB2 and PGE2 produced Whole blood was obtained with a 19-gauge needle from healthy volunteers by cultured PBMC were measured in the supernatant by enzyme immu- who had not taken aspirin or other nonsteroidal anti-inflammatory drugs in noassays (Cayman). the 7 days preceding the donation. Blood was anticoagulated with triso- dium citrate (0.129 M; Becton Dickinson, Meylan, France) and centrifuged Statistical analysis at 280 3 g for 15 min at 4°C. Platelet-rich plasma was removed. The All results represent the mean 6 1 SEM of 4 to 10 separate experiments. sedimented cells were diluted to twice the original blood volume with PBS Depending on the data, statistical analysis was performed using Student’s (pH 7.4; Seromed, Biochrom, Berlin, Germany), layered onto Ficoll- test for paired variables or ANOVA followed by a Neuman-Keuls test Hypaque PLUS (Pharmacia Biotech, Uppsala, Sweden), and centrifuged at when p # 0.05. Differences were