1 Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2 Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more eective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the eects of trans-resveratrol (3,4',5-trihydroxytrans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4 trans-Resveratrol exerted a strong inhibitory eect on reactive oxygen species produced by PMN stimulated with 1 mM formyl methionyl leucyl phenylalamine (fMLP) (IC 50 1.3+0.13 mM, mean+ s.e.mean), as evaluated by luminol-ampli®ed chemiluminescence. 5 trans-Resveratrol prevented the release of elastase and b-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 mM, IC 50 18.4+1.8 and 31+1.8 mM), and C5a (0.1 mM, IC 50 41.6+3.5 and 42+8.3 mM), and also inhibited elastase and b-glucuronidase secretion (IC 50 37.7+7 and 25.4+2.2 mM) and production of 5-lipoxygenase metabolites leukotriene B 4 (LTB 4 ), 6-trans-LTB 4 and 12-trans-epi-LTB 4 (IC 50 48+7 mM) by PMN stimulated with the calcium ionophore A23187 (5 mM). 6 trans-Resveratrol signi®cantly reduced the expression and activation of the b 2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated ®xed platelets in a dynamic system were also prevented by transresveratrol. 7 These results, indicating that trans-resveratrol interferes with the release of in¯ammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective eect of red wine consumption against CHD.

SummaryIn PMN/platelet suspensions stimulated by fMLP giant mixed aggregates are formed and TxB2 and LTC4 are synthesized as the result of the cooperation in the arachidonic acid (AA) metabolism during cell/cell contact. PMN-derived cathepsin G induced the expression of P-selectin on platelet surface. GE12, an antibody against P-selectin, significantly reduced mixed cell aggregates. GE12 did not affect platelet aggregation induced by PMN-derived supernatants, indicating that the inhibitory effect of GE12 on mixed cell aggregation depends on inhibition of PMN/platelet adhesion. GE12 significantly reduced TxB2 and LTC4 production in PMN/platelet mixed cell suspensions stimulated by fMLP. As previously reported, synthesis of 3H-TxB2 in 3H-AA-labeled PMN/unlabeled platelets indicates that platelets utilize 3H-AA from PMN. 3H-LTC4 production in unlabeled PMN/3H-AA-labeled platelets indicates that bidirectional routes are utilized in this system for LTC4 synthesis. GE12 significantly reduced 3H-TxB2 and 3H-LTC4 synthesis. These results show that cathepsin G released by activated PMN induces the expression of P-selectin on platelet membrane: this adhesive glycoprotein modulates cell-cell contact and transcellular metabolism of AA.

Human polymorphonuclear leukocytes (PMN) activated by n-formyl- methionyl-leucyl-phenylalanine (fMLP), in the presence of cytochalasin B, are able to induce activation of coincubated autologous platelets “via” cathepsin G released from the azurophilic granules. However, thromboxane (Tx) B2 production in this system cannot be completely explained by cathepsin G-stimulated platelet arachidonate metabolism. Indeed, the amount of TxB2 found in supernatants of platelet/PMN suspensions challenged with 1 mumol/L fMLP was twofold to fourfold higher than that measured when platelets were stimulated by supernatants from fMLP-activated PMN. In the present report, we analyzed the possibility that PMN-induced TxB2 production in this system is the result of transcellular metabolism of arachidonic acid (AA) between fMLP-activated PMN and cathepsin G-stimulated platelets. 3H-AA-labeled PMN were used to test if a transfer of AA or metabolite(s) occur from PMN to platelets. Our results showed that: (1) 3H-TxB2 and 3H-12-HHT are synthesized when 3H-AA-labeled PMN are activated mixed to unlabeled platelets; (2) total radioactivity released by fMLP-stimulated PMN is increased in the presence of platelets, whereas the membrane content of unesterified 3H-AA is reduced; (3) platelet cyclooxygenase inhibition completely prevents 3H- TxB2 synthesis; and (4) inhibition of cathepsin G-induced platelet activation with the antiprotease eglin C blocks the formation of 3H- TxB2. These data show that in the experimental system used, platelets use PMN-derived unmetabolized AA to synthesize TxB2.

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