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.

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.

Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD11b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + 2, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, 2 hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation.

Polymorphonuclear leukocyte (PMN)-platelet interactions at sites of vascular damage contribute to local and systemic inflammation. We sought to determine the role of "outside-in" signaling by Srcfamily tyrosine kinases (SFKs) in the regulation of ␣M␤2-integrin-dependent PMN recruitment by activated platelets under (patho)physiologic conditions. Activationdependent epitopes in ␤2 integrin were exposed at the contact sites between PMNs and platelets and were abolished by SFK inhibitors. PMNs from ␣M␤2 ؊/؊ , hck ؊/؊ fgr ؊/؊ , and hck ؊/؊ fgr ؊/؊ lyn ؊/؊ mice had an impaired capacity to adhere with activated platelets in suspension. Phosphorylation of Pyk2 accompanied PMN adhesion to platelets and was blocked by