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Platelets Abrogate Leukotriene B4 Generation by Human Blood 0023-6837/03/8304-491$03.00/0 LABORATORY INVESTIGATION Vol. 83, No. 4, p. 491, 2003 Copyright © 2003 by The United States and Canadian Academy of Pathology, Inc. Printed in U.S.A. Platelets Abrogate Leukotriene B4 Generation by Human Blood Neutrophils Stimulated with Monosodium Urate Monohydrate or f-Met-Leu-Phe In Vitro Bernard Chabannes,† Patrice E. Poubelle, Patrick Molière, Rinaldo De Médicis, André Lussier, and Michel Lagarde INSERM U.352, Laboratoire de Biochimie et Pharmacologie INSA-Lyon (BC, PEP, PM, ML), Villeurbanne, France; and Centre de Recherche en Rhumatologie et Immunologie and the Department of Medicine (PEP), Université Laval, Que´ bec, and Faculty of Medicine (RDM, AL), Sherbrooke University, Sherbrooke, Québec, Canada SUMMARY: Neutrophils are physiologically associated with platelets in whole blood. Inflammatory reactions can be modulated by the presence of platelets. To investigate the influence of platelets on neutrophil activity, we studied the 5-lipoxygenase (5-LOX) metabolic pathway in normal human blood neutrophils stimulated with f-Met-Leu-Phe (fMLP) or monosodium urate monohydrate (MSUM) in the presence of autologous platelets. Platelets inhibited by more than 90% the synthesis of leukotriene B4 and 5-HETE in neutrophils activated with fMLP or MSUM. The addition of exogenous arachidonic acid did not reverse the inhibitory effect of platelets on 5-LOX–generated metabolites in fMLP- or MSUM-activated neutrophils. Preincubation of neutrophils with adenosine deaminase reversed the inhibitory effect of platelets in fMLP-treated neutrophils, indicating that adenosine was responsible for the platelet inhibition of leukotriene B4 and 5-HETE formation. In contrast, adenosine deaminase had no influence on the inhibitory effects of platelets in MSUM-stimulated cells. These results suggest that platelets can inhibit the synthesis of 5-LOX products (a) by acting mainly downstream to phospholipase A2 in cells stimulated by fMLP or MSUM, (b) through adenosine when neutrophils are activated with fMLP, and (c) by an adenosine-independent mechanism in MSUM-activated neutrophils by an as-yet- unidentified mediator. (Lab Invest 2003, 83:491–499). eukotriene B4 (LTB4) release leads to major bio- HPETE) (Rouzer et al, 1985). This intermediate metab- logic consequences associated with inflamma- olite is further converted into LTA by 5-LOX and into L 4 tion. This arachidonic acid (AA) metabolite is one of 5-HETE by glutathione peroxidase. Since its discovery the most potent inflammatory chemoattractants of in 1979 (Borgeat and Samuelsson, 1979), LTB4 and polymorphonuclear and mononuclear phagocytes the other bioactive 5-LOX–derived products have (Ford-Hutchinson, 1990). Besides neutrophil chemo- been the focus of an intense research to obtain 5-LOX taxis and aggregation, LTB4 induces neutrophil de- and FLAP inhibitors and more specifically LTB4 antag- granulation and lysosomal enzyme release (Sha’afi et onists. Neutrophils also have the capacity to metabo- al, 1981) and neutrophil endothelial cell adhesion lize LTB4 directly into a less active mediator 20-OH- (Hoover et al, 1984), is involved in immune modulation LTB4 and an inactive product 20-COOH-LTB4. (Henderson, 1994; Poubelle et al, 1991), and mediates Deposits of platelets in inflamed tissues during neu- inflammatory pain (Levine et al, 1984). LTB4 is gener- trophil margination are associated with cell-to-cell inter- ated through a cytosolic LTA4-hydrolase (Radmark et actions and modulation of the inflammatory process al, 1984). The unstable compound LTA4 is formed (Issekutz et al, 1983). Platelets and neutrophils exert through the action of 5-lipoxygenase (5-LOX) on AA in multiple and complex functions in pathophysiologic con- the presence of a 5-LOX–activating protein (FLAP), ditions of inflammation (Poubelle and Borgeat, 2002). leading to 5-hydroperoxyeicosatetraenoic acid (5- Activated platelets adhere to neutrophils (Jungi et al, 1986) through their ␣-granule membrane glycoprotein GMP-140 (or P-selectin) (Larsen et al, 1989) and the integrin complex GPIIb/IIIa-fibrinogen (Spangenberg et DOI: 10.1097/01.LAB.0000062855.90029.D8 al, 1993). Aggregates of platelets and neutrophils lead to Received November 18, 2002. metabolic or functional interactions between these cells †Deceased. This work was supported by INSERM (France), the Région (Ginsburg and Quie, 1980; Marcus et al, 1982; McGarrity Rhône-Alpes, the FRSQ (Québec, Canada) via a Programme de Coopéra- tion (no 980971) and the Université Laval (sabbatical year to PEP). et al, 1988; Weksler, 1983). For instance, LTA4 from Address reprint requests to: Dr. Patrice E. Poubelle, Centre de Recherche du neutrophils is metabolized by platelets into LTC4 (Ma- CHUL, 2705 boul Laurier, Ste-Foy, Qué, G1V 4G2, Canada. E-mail: clouf et al, 1989) and lipoxins (Serhan and Sheppard, [email protected] 1990). However, 12-HETE from platelets is converted to Laboratory Investigation • April 2003 • Volume 83 • Number 4 491 Chabannes et al 12,20-diHETE by unstimulated neutrophils (Marcus et al, by HPLC, all of the experiments were conducted in 1984). Platelets activated with thrombin can increase conditions in which the available endogenous sub- LTB4 synthesis by neutrophils in the presence of zymo- strate AA could be increased. Therefore, studies of the san, f-Met-Leu-Phe (fMLP), C5a, or PAF (Palmantier and effects of fMLP were carried out with neutrophils Borgeat, 1991). Conversely, co-incubation of washed pretreated with thimerosal to inhibit the reacylation of platelets with neutrophils activated by the nonphysi- AA into phospholipids. In these conditions, the ologic stimulus ionophore A23187 is associated with an 5-lipoxygenase products measured were previously increase of 12-HETE and a decrease of 5-HETE, LTB4, reported to be approximately 200 pmol/ml of incubate and thromboxane B2 (Maderna et al, 1993). In addition, compared with 1000 pmol/ml when neutrophils were neutrophils can inhibit the AA metabolism in platelets stimulated by 0.5 ␮M ionophore A23187 in the ab- stimulated with thrombin, collagen, or ionophore (Cha- sence of thimerosal (Chabannes et al, 1997). Studies bannes et al, 1994). Platelets from healthy subjects also of the effects of MSUM crystals were performed with have the capacity to modulate neutrophil superoxide neutrophils primed by GM-CSF, a factor that has been anion production and chemiluminescence depending on reported to increase the production of leukotrienes the stimulus used in vitro (Carulli et al, 1995; Colli et al, induced by phagocytosis (Poubelle et al, 1989) 1996). However, platelets from uremic or allergic patients through an enhancement of the release of AA (DiPer- are altered and are unable to decrease the production of sio et al, 1988). Note that despite a pretreatment of superoxide anion (Carulli et al, 1995) or LTB4 by A23187- neutrophils with thimerosal or GM-CSF, platelets were activated neutrophils (Hosni et al, 1991). Circulating shown to exert a significant effect on AA metabolism platelets bind to neutrophils, and platelet-neutrophil in neutrophils activated by a soluble or a particulate complexes have been demonstrated in whole blood agonist (see below). It is also useful to stress that (Rinder et al, 1991). Such a satellitism, already described MSUM, unlike fMLP, can stimulate platelets to release in 1974 (Kjeldsberg and Swanson, 1974), may have AA (Serhan et al, 1984). potent pathophysiologic relevance because at rest, 25% Figure 1 shows the amounts of LTA4-derived prod- ϩ ϩ of blood neutrophils are complexed with platelets, and ucts (LTB4 20-OH-LTB4 trans-isomers of LTB4) after platelet activation with ADP or thrombin, such and 5-HETE synthesized by neutrophils (N) stimulated complexes rise to 70% (Peters et al, 1997). Besides their with 1 ␮M fMLP in the absence or in the presence of phlogogenic activity through interactions with neutro- autologous platelets (P) with a ratio of P/N ϭ 30/1. phils, platelets can counterbalance neutrophil activation. Inhibition of the synthesis of 5-LOX–derived products Platelets from healthy donors can reduce LTB4 synthesis was correlated to platelet concentration and was in A23187-stimulated neutrophils from allergics (Hosni et almost complete (80% to 90% inhibition) at a ratio of al, 1991). Thus, unactivated platelets might act as a 30P/1N; a 50% inhibition was recorded at 10P/1N (not natural inhibitor of LTB4 formation by neutrophils in diseases such as bacterial infection or arthritis. To ad- dress this question, we investigated the effects of normal human blood platelets on the 5-LOX activity of autolo- gous neutrophils stimulated with the pathophysiologic stimuli fMLP, a biologically active ligand produced by bacteria, or monosodium urate monohydrate (MSUM) crystals, the causal agent of gout. The results of the present study demonstrate that platelets have the ca- pacity to abrogate the synthesis of LTB4 and 5-HETE in fMLP- and MSUM-activated neutrophils. The inhibitory influence of platelets on the synthesis of LTB4 and 5-HETE was not reversed by the addition of exogenous AA, suggesting an effect of platelets downstream to the neutrophil phospholipase A2 (PLA2). A candidate to this platelet inhibitory factor could be adenosine, an endog- enous product that down-regulates ligand-stimulated LTB4 biosynthesis in neutrophil suspensions (Krump et al, 1997). As suspected, adenosine was the platelet inhibitory factor of LTB4 and 5-HETE generated by fMLP-activated neutrophils, whereas platelet inhibition of MSUM-stimulated neutrophils was independent of Figure 1. adenosine. Effect of autologous platelets on 5-LOX–derived products generation by human blood neutrophils stimulated by fMLP. Neutrophils preincubated with ␮ Results thimerosal were further incubated with 1 M fMLP in the absence or presence of platelets (platelet/neutrophil ratio: 30/1) for 5 minutes at 37° C. LTA4- ϩ ϩ derived products (LTB4 20-OH-LTB4 trans-isomers of LTB4) and 5-HETE Effects of Autologous Platelets on LTB4 and 5-HETE were measured by RP-HPLC, and amounts are expressed in picomoles formed 7 Synthesis by Neutrophils by 10 neutrophils/ml. Results are the mean Ϯ SEM of 20 separate experiments carried out in duplicate.
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