The CC chemokines MDC and TARC induce platelet activation via CCR4.

While chemokines have received considerable attention for their role in leukocyte chemotaxis, their effects on platelets have not been well described. We found that two CC chemokine receptor 4 (CCR4) ligands, macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC) induce concentration-dependent platelet aggregation and calcium flux. Flow cytometric analysis revealed the expression of CCR4 on platelets and a monoclonal antibody (mAb) to CCR4 inhibited MDC- and TARC-induced platelet aggregation, confirming that this effect is mediated through their common receptor CCR4. MDC fully desensitized TARC-induced calcium mobilization in platelets, while TARC was unable to completely desensitize a subsequent MDC response, which is similar to observations made in Th2 CD4(+) lymphocytes and CCR4-transfected cells. Aspirin (ASA) treatment of platelets allowed reversible primary aggregation but inhibited irreversible complete aggregation, suggesting that MDC- and TARC-induced full platelet aggregation is dependent on cyclooxygenase metabolites of arachidonic acid. MDC and TARC were unable to induce platelet aggregation and platelet secretion in washed human platelets, even though they induced a calcium flux, suggesting that plasma components are required for MDC- and TARC-induced platelet aggregation. Since Th2-type cytokines induce the release of MDC and TARC from cells and the expression of these chemokines is increased in Th2-type inflammation, we hypothesize that MDC and TARC may play a role in platelet activation seen in Th2 diseases, such as asthma and atopic dermatitis.

Monocyte chemoattractant protein (MCP)-4 expression in the airways of patients with asthma. Induction in epithelial cells and mononuclear cells by proinflammatory cytokines.

Chemokines are chemotactic cytokines that play an important role in recruiting leukocytes in allergic inflammation. Monocyte chemoacctractant protein (MCP)-4 is a CC chemokine with potent chemotactic activities for eosinophils, monocytes, T lymphocytes, and basophils and therefore represents a good candidate to participate in allergic reactions. To determine if MCP-4 plays a role in asthma, we have investigated the expression of MCP-4 messenger RNA (mRNA) and protein in the airways of patients with asthma and normal control subjects by in situ hybridization and immunohistochemistry. We found that MCP-4 mRNA and protein was significantly upregulated in the epithelium and submucosa of bronchial biopsies and in the bronchoalveolar lavage (BAL) cells of patients with asthma compared with normal control subjects (p < 0. 01). In addition, MCP-4 protein was significantly elevated in the BAL fluid of patients with atopic asthma when compared with normal control subjects (p < 0.01) and there was a significant correlation between MCP-4, eotaxin, and eosinophils. In support of our in situ findings demonstrating MCP-4 expression in epithelial cells and mononuclear cells in vivo, we have found that MCP-4 expression can be induced in these cells in vitro by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Interferon-gamma (IFN-gamma) acted synergistically with TNF-alpha and IL-1beta in the induction of mRNA MCP-4 mRNA expression in A549 cells, whereas the glucocorticoid dexamethasone diminished the cytokine-induced expression of MCP-4. Our findings demonstrate that MCP-4 is upregulated in the airways of patients with asthma and suggest that MCP-4 plays a role in the recruitment of eosinophils into the airways of patients with asthma.

The stromal cell-derived factor-1 chemokine is a potent platelet agonist highly expressed in atherosclerotic plaques.

Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes. However, their role in modulating platelet function has not been shown. We studied the direct effect of chemokines on human platelets and found that of the 16 tested only stromal cell-derived factor (SDF)-1 induced platelet aggregation, accompanied by a rise in intracellular calcium. Platelets expressed the SDF-1 receptor, CXCR4, and an antibody to CXCR4 and pertussis toxin inhibited SDF-1-induced platelet aggregation, confirming that this effect is mediated through CXCR4, a Galphai-coupled receptor. SDF-1-induced platelet aggregation was also inhibited by wortmannin, LY294002, and genistein, suggesting that phosphatidylinositol 3-kinase and tyrosine kinase are likely involved in SDF-1-induced platelet aggregation. Because chemokines are produced from multiple vascular cells and atherosclerotic vessels are prone to develop platelet-rich thrombi, we examined the expression of SDF-1 in human atheroma. SDF-1 protein was highly expressed in smooth muscle cells, endothelial cells, and macrophages in human atherosclerotic plaques but not in normal vessels. Our studies demonstrate a direct effect of a chemokine in inducing platelet activation and suggest a role for SDF-1 in the pathogenesis of atherosclerosis and thrombo-occlusive diseases.

Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation.

Presently, there is considerable evidence for the participation of eosinophils in the pathophysiology of human bronchial asthma. Although increased numbers of eosinophils are present in the airways and bronchoalveolar lavage (BAL) fluid of atopic asthmatics, the mechanisms responsible for their preferential accumulation are still largely unknown. Eotaxin is a chemokine that promotes the selective recruitment of eosinophils. We report that atopic asthmatic patients have high concentrations of eotaxin in BAL fluid and an increased expression of eotaxin mRNA and protein in the epithelium and submucosa of their airways when compared with normal controls. In the BAL cells from asthmatic patients, eotaxin immunoreactivity colocalized predominantly to macrophages (62.2%), with a lesser contribution from T cells (16.3%) and eosinophils (8.9%). BAL fluid from asthmatics contained chemotactic activity for eosinophils that was attributable in part to the presence of eotaxin. Moreover, eotaxin was more effective at inducing in vitro eosinophil chemotaxis when eosinophils were stimulated with IL-5 (a cytokine that enhances the effector capacity of mature eosinophils). These observations suggest that eotaxin contributes to the pathogenesis of asthma by the specific recruitment of eosinophils into the airways.

Non-genomic effects of estrogen and the vessel wall.

Estrogen, like other steroids, is now believed to possess rapid membrane effects independent of the classical gene activation pathway of steroid action. The presence of membrane estrogen receptors has been demonstrated in different cell types, but not yet in vascular tissue. In vivo, estrogen administration rapidly promotes acetylcholine-induced vasodilation of the coronary and peripheral vascular beds of postmenopausal women. Estrogen also causes relaxation of precontracted isolated arterial segments and perfused organ preparations, within minutes of administration of the hormone. These rapid vasomotor effects of estrogen may be related to blockade of the cell membrane voltage-dependent calcium channels, resulting in inhibition of extracellular Ca2+ mobilization and flux. Recently, estradiol has been shown to rapidly affect cyclic nucleotide turnover in vascular segments, smooth muscle, and epithelial cell cultures, suggesting the possibility of a "cross-talk" between membrane-mediated events and nuclear receptor activation.

Estradiol increases cyclic adenosine monophosphate in rat pulmonary vascular smooth muscle cells by a nongenomic mechanism.

Estrogen, like other steroids, may induce rapid nongenomic cellular effects. We studied the effect on intracellular cAMP of short-term exposure (5 min) of cultured rat pulmonary vascular smooth muscle cells (VSMC) to estradiol 17 beta. At confluence, VSMC were incubated in phosphate buffer saline for 1 hr before exposure to different hormones. The reaction was stopped with 0.1 N HCl and cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The 5-min incubation with estradiol 17 beta (0.3-30 microM) significantly increased basal intracellular cAMP in a concentration-dependent manner. The stimulatory effect of estradiol on cAMP was time-dependent, increasing with prolonged exposure to the hormone, and was not affected by the protein synthesis inhibitor, actinomycin D (5 micrograms/ml), at 5 and 30 min. Comparable concentrations of testosterone or estradiol 17 alpha had no significant effect on cAMP. The estrogen receptor partial agonist, tamoxifen also significantly increased basal cAMP in a concentration-dependent manner, but inhibited the effect of estradiol. Furthermore, forskolin elicited a concentration-dependent increase in cAMP (396.6 +/- 53% at 10 microM concentration), which was significantly potentiated in presence of estradiol. The effect of estradiol is unlikely to be mediated by G-protein activation, because the G protein inhibitor, pertussis toxin (100 ng/ml), did not significantly affect estradiol-induced increase in cAMP. Removal of Ca++ from the incubation medium inhibited the stimulatory effect of estradiol 17 beta suggesting that estradiol may increase pulmonary VSMC cAMP via a Ca(++)-dependent pathway. We suggest that the effect of estradiol 17 beta in these experiments is nongenomic in nature, and is possibly mediated by direct interaction of the hormone with specific membrane binding sites.

Inhibition of platelet aggregation in whole blood by alcohol.

Our previous studies have demonstrated that addition of moderate volumes of absolute alcohol (34-170 mM final concentration) to whole blood produces concentration-dependent platelet aggregation, due to release of adenosine diphosphate (ADP) from erythrocytes. We have now investigated the effects of exposure of blood to ethanol by a more "physiologic" protocol, in which 7.8% (w/v) alcohol is added to achieve a final concentration of 1 to 85 mM in human and rat blood or platelet rich plasma (PRP). The effects of short incubation with alcohol on platelet aggregation induced by ADP, collagen and arachidonic acid were examined by the impedance method of aggregometry. Aggregation induced by collagen in PRP of either species was significantly inhibited by 85 mM ethanol, with concentrations as low as 4.25 mM inhibiting the response to collagen in rat whole blood. ADP stimulated only primary, reversible aggregation in rat PRP and whole blood, and these responses were unaffected by alcohol. Human platelets responded to ADP with irreversible aggregation, which was significantly attenuated by 85 mM ethanol in whole blood but not PRP. Arachidonic acid evoked irreversible platelet aggregation in all four preparations; this was significantly inhibited by the high dose ethanol in human and rat PRP, but not whole blood. In contrast to our earlier studies with absolute ethanol, there was no evidence of hemolysis (and therefore, ADP release from red blood cells) using the current protocol. The results of these experiments show that alcohol, at physiologically relevant concentrations, has an inhibitory effect on secondary platelet aggregation responses to some agonists in whole blood as well as PRP, possibly by its previously demonstrated effects on arachidonic acid release by phospholipases. The possibility remains to be considered that other blood cells might contribute to the effects of alcohol on platelet aggregation in whole blood.

Mechanism of ethanol-induced aggregation in whole blood.

Effects of ethanol on blood clotting and platelet aggregation have been reported in many models, but its in vitro actions in whole blood, impedance aggregometry have not been reported. We investigated the effect of ethanol in vitro in whole blood and platelet rich plasma of humans and rats, as measured by impedance aggregometry. Ethanol (34 to 170 mM) induced concentration-dependent aggregation in whole blood but not platelet rich plasma. In further studies in rats, aggregation was inhibited by pretreatment of whole blood with the prostacyclin analog iloprost or the enzyme apyrase, which degrades ADP to AMP. Levels of ethanol which produced aggregation in whole blood were also associated with concentration-dependent hemolysis. Based on the requirement for whole blood for ethanol-induced aggregation, the inhibitory effect of apyrase and our observation of hemolysis, and previous studies which have demonstrated the potential contribution of ADP from lysed red blood cells to platelet aggregation, we conclude that ethanol-induced aggregation in whole blood is mediated by erythrocyte lysis and the ADP released from these cells.

Re-evaluation of the effects of neuropeptide Y on aggregation of human platelets.

Several studies have shown that platelets are a major source of circulating neuropeptide Y (NPY) immunoreactivity in rats, but the effects of this vasoconstrictor peptide on platelets are not well-defined. Recently, it was reported that porcine NPY was an inhibitor of in vitro human platelet aggregation induced by epinephrine, an observation which would have important implications regarding platelet-vascular interactions during states involving platelet activation and thrombosis. Thus, we undertook the present studies, in an attempt to confirm the earlier report, and to extend those observations to human NPY. In contrast to the recent report, we found no inhibitory effect of either human or porcine NPY on epinephrine- or collagen-induced aggregation of human platelets from normal subjects. Likewise, specific NPY Y-1 and Y-2 agonists had no direct or indirect action on platelet aggregation. Finally, the effect of human NPY on intraplatelet cAMP was measured. The peptide had no effect on either basal or iloprost-stimulated cAMP levels. We hypothesize that the role of NPY in the platelet-vascular interaction is in promoting vasoconstriction associated with platelet aggregation, and does not include inhibition of further thrombosis.