ABSTRACT
Interleukin-8 (IL-8) is generally accepted to be an important mediator of a number of acute and chronic inflammatory diseases and is produced by monocytes upon stimulation by lipopolysaccharide (LPS). Epinephrine has been reported by several groups to suppress activation of monocytes in response to LPS, and the aim of the present study was to examine the effect of epinephrine on LPS induced IL-8 production using whole blood as a model system. Epinephrine increased LPS induced IL-8 production in a dose-dependent manner in the whole concentration range (0.001-100 microM) and 1 microM epinephrine increased IL-8 levels with 125%. Epinephrine per se had no effect on IL-8 levels. The potentiating effect of epinephrine was mediated by blood platelets, since IL-8 levels in samples containing platelets and stimulated with LPS and epinephrine (1-100 microM) were significantly higher (p<0.05) than in control samples containing no platelets. This effect of platelets seemed to be due to platelet release products, since addition of 25 microL platelet lysate supernatant to whole blood increased LPS induced IL-8 production with 100% and a similar effect was observed in freshly isolated mononuclear cells resuspended in plasma. Upon addition of 50 microg/ml of the carboxyterminal peptide of platelet factor 4 (PF4(58-70)) to whole blood, LPS stimulated IL-8 levels were increased with 115%, whereas in mononuclear cells, 20 microg/ml PF4(58-70) enhanced IL-8 production with 40%. We demonstrate for the first time that epinephrine promotes LPS induced production of IL-8 in whole blood via an effect on blood platelets. This potentiating effect of platelets is shown to be due to platelet granule contents, and platelet factor 4 (PF4) is suggested to be one of several platelet granule proteins promoting LPS induced IL-8 production in whole blood.
Subject(s)
Blood Platelets/physiology , Epinephrine/pharmacology , Interleukin-8/blood , Vasoconstrictor Agents/pharmacology , Humans , Lipopolysaccharides/pharmacology , Platelet Activation , Platelet Factor 4/metabolismABSTRACT
Several components of blood, e.g. lipids, coagulation and fibrinolytic factors, are thought to be important risk factors in cardiovascular diseases. The aim of this study was to correlate these risk factors and the soluble adhesion proteins, soluble P-selection (sP-selectin) and soluble vascular cell adhesion molecule (sVCAM-1), in healthy men and women as well as to unravel any effects of smoking. One hundred and forty-two fasting men (median age 36 years) including 39 smokers, and 124 women (median age 34 years) including 35 smokers, were tested between 0800 h and 1000 h. Fibrinogen correlated positively with white blood cells (WBC) (r = 0.25), prothrombin fragment 1.2 (F1.2) (r = 0.21), cholesterol (r = 0.27), beta-thromboglobulin (r = 0.29), Factor VII clotting activity (FVIIc) (r = 0.27) (all P < 0.0001), tissue plasminogen activator antigen (t-PAag) (r = 0.22, P < 0.0005), plasminogen activator inhibitor-1 antigen (PAI-1ag) (r= 0.20) and VCAM-1 (r= 0.19) (both P< 0.002). Cholesterol and triacylglycerol (TG) correlated positively with t-PA antigen (t-PAag) (r = 0.36 and r = 0.38), PAI-1 antigen (PAI-1ag) (r = 0.35 and r = 0.50), P-selectin (r = 0.26 and r = 0.27) (all P < 0.0001) and WBC (r = 0.17, P < 0.007 and r = 0.18, P < 0.004). Cholesterol correlated also with F1.2 (r = 0.29) and TG (r= 0.44) (P< 0.0001). In addition to cholesterol and TG, sP-selectin correlated postively with PAI-1ag (r= 0.39), t-PAag (r= 0.27) and WBC (r = 0.25) (all P < 0.0001). Comparing the various test parameters in men and women, it was found that women had significantly higher levels of F 1.2 and high-density lipoprotein-cholesterol than men, whereas men had higher levels of t-PAag, PAI-lag and P-selectin than women. Smoking was associated with a rise in several of the test parameters. It can be concluded that there are correlations between several risk factors. Of particular interest is the positive correlation between sP-selectin and a number of established risk factors of cardiovascular diseases.
Subject(s)
Hemostatics/blood , Lipids/blood , P-Selectin/blood , Vascular Cell Adhesion Molecule-1/blood , Adult , Age Factors , Antigens/blood , Biomarkers/blood , Blood Coagulation Factors/analysis , Cardiovascular Diseases/etiology , Cholesterol/blood , Female , Fibrinogen/analysis , Humans , Leukocyte Count , Lipids/chemistry , Male , Plasminogen Activator Inhibitor 1/blood , Plasminogen Activator Inhibitor 1/immunology , Premenopause , Risk Factors , Smoking/adverse effects , Tissue Plasminogen Activator/blood , Tissue Plasminogen Activator/immunology , beta-Thromboglobulin/analysisABSTRACT
In the past years, our group has made several observations suggesting that blood cells behave differently and when stimulated, release different levels of cytokines, depending on which anticoagulant the blood has been drawn into. The aim of this study was therefore to compare the effect of the four anticoagulants EDTA, citrate, heparin and hirudin on monocyte, neutrophil (PMN), and platelet function in human whole blood. Human whole blood was employed as an ex vivo model of cytokine production and protein secretion, and lipopolysaccharide (LPS) induced tissue factor (TF) activity in monocytes and LPS induced tumor necrosis factor alpha (TNF alpha) release were chosen as parameters of monocyte activation. Platelet factor 4 (PF4) secretion and LPS induced lactoferrin release were chosen as parameters of platelet and PMN activation, respectively. When human whole blood was stimulated with 5 ng/ml LPS for 2 h, TF activity in monocytes isolated from EDTA blood was found to be 2.9 mU/10(6) cells, whereas TF activity in monocytes isolated from citrated, heparinized and hirudinized blood was 14.7, 24.7 and 28.5 mU/10(6) cells, respectively. TNF alpha concentrations in platelet poor plasma (PPP) isolated from whole blood stimulated with 5 ng/ml LPS for 2 h was increased with 200, 400 and 350% in citrated, heparinized and hirudinized blood respectively, as compared to EDTA blood. Next, the effect of the anticoagulant on PMN secretion was measured. PPP isolated from whole blood incubated with 5 ng/ml LPS for 90 min contained 1170 ng/ml (EDTA blood), 2880 ng/ml (citrated blood), 4220 ng/ml (heparinized blood), and 5520 ng/ml lactoferrin (hirudinized blood). When studying the platelet parameter PF4, whole blood was incubated without any stimuli for 60 min, and we found that heparin PPP contained 1180 ng/ml PF4, while hirudin PPP contained 469 ng/ml, citrate PPP 440 ng/ml, and EDTA PPP 217 ng/ml PF4, respectively. Finally, the low molecular weight heparin compound Fragmin had no enhancing effect on PF4 levels in whole blood. It is concluded that the anticoagulant used in in vitro experiments has a large influence on the parameters measured.
Subject(s)
Anticoagulants/pharmacology , Monocytes/drug effects , Neutrophil Activation/drug effects , Platelet Activation/drug effects , Citric Acid/pharmacology , Drug Evaluation , Edetic Acid/pharmacology , Heparin/pharmacology , Hirudins/pharmacology , Humans , Lipopolysaccharides/pharmacology , Reference ValuesABSTRACT
In a previous study we have shown that granulocytes enhance lipopolysaccharide (LPS)-induced tissue factor (TF) activity in monocytes in a platelet-dependent reaction. The present investigation was undertaken to examine the role of a platelet activation product, platelet factor 4 (PF4), in LPS-induced TF activity in monocytes. Platelet lysate supernatant, purified PF4, and the COOH-terminal tridecapeptide of PF4, termed PF4(58-70), enhanced LPS-induced TF activity in monocytes of whole blood dose dependently. A monoclonal antibody against P-selectin eliminated the enhancing effect of PF4(58-70) on LPS-induced TF activity in monocytes, and PF4(58-70) was shown to act synergistically with tumor necrosis factor alpha (TNF-alpha). However, PF4(58-70) did not enhance TNF-alpha secretion in LPS-stimulated whole blood. The major effect of PF4(58-70) was granulocyte dependent. Our results suggest that PF4 might play an important role in LPS-stimulated monocyte TF activity of whole blood.