Pro- and anti-inflammatory effects of histamine on tissue factor and TNFα expression in monocytes of human blood.

BACKGROUND: Histamine is classified as an inflammatory mediator and has been reported to have anti- as well as pro-inflammatory properties. The aim of this study was to explore the role of histamine on the production of LPS-induced tissue factor (TF) activity and TNFα in monocytes of whole blood in the absence and presence of TNFα or PMA. METHODS: Human blood anticoagulated with Fragmin was subjected to stimulation by LPS in the presence and absence of TNFα or PMA and various concentrations of histamine. Tissue factor (TF) activity was measured in lyzed cells after isolation of mononuclear cells whereas TNFα was quantified in plasma after centrifugation of cells. RESULTS: Histamine gave a dose dependent inhibitory effect on LPS-induced TF activity in monocytes of whole blood, with a 50% reduction at 0.033 µM. A similar effect was seen when the blood cells were stimulated with the combination of LPS and TNFα although TNFα enhanced LPS-induced TF activity almost two fold. In contrast, when blood was incubated with LPS and PMA in whole blood, histamine gave a significant rise in TF activity at 0.01 µM and 0.33 µM histamine. The effect of histamine was less at 0.1 µM or higher concentrations giving a biphasic profile. Contrary to the effect of histamine on LPS plus PMA induced TF activity, histamine caused a significant reduction in TNFα albeit less than in the absence of PMA. Intake of aspirin caused a significant rise in LPS-induced TF activity that was almost abolished by histamine at 0.033 µM. CONCLUSION: Our study shows that histamine has an anti-inflammatory effect on LPS and LPS/TNFα stimulated monocytes of whole blood. In contrast when blood cells are activated by a combination of LPS and PMA whereby PKC is activated, histamine has a procoagulant/pro-inflammatory effect through enhancement of TF activity expression.

Human platelets do not express tissue factor.

BACKGROUND: The controversy about the expression of tissue factor (TF) in platelet after de novo synthesis prevail despite many groups recognize that platelet isolation, assays and reagents, particularly non-specific antibodies, may account for the diversity. In this study the potential of TF expression was evaluated using immune-purified human platelets and employing a very sensitive and highly specific TF activity assay. METHODS: Isolated platelets in plasma anti-coagulated with Fragmin were subjected to stimulation by LPS plus PMA, IgG antibody or TRAP and tested for TF activity. RESULTS: Platelets stimulated with LPS plus PMA for 4 hours expressed trace amounts of TF like activity (PCA), not inhibited by anti-TF antibody (0.2±0.1 mU/ml blood). Platelets, not immune-adsorbed to remove monocytes, showed significant TF activity (2.0±0.9 mU/ml blood) that was nearly abolished by anti-TF antibody. IgG antibody from patient with lupus anticoagulant failed to enhance the trace amount of PCA as compared to the control in contrast to high TF activity induced in monocytes (0.4±0.1 mU/ml blood versus 27.5±10.5 mU/10(6) cells) showing that activation of complement is not mediating TF expression. Platelet subjected to TRAP activation for 10 min possessed only trace amounts of PCA that was not inhibited by anti-TF antibody and slightly enhanced by anti-TFPI antibody. CONCLUSIONS: It is concluded that platelets free of monocytes do not express TF activity when stimulated by LPS or activated complement factors, implying no role for Toll like receptor (TLR4) as suggested recently. There is no evidence of TF activity associated with platelets as a result of rapid and dynamic process.

Differential ability of tissue factor antibody clones on detection of tissue factor in blood cells and microparticles.

INTRODUCTION: Tissue factor (TF), the primary initiator of coagulation in vivo, plays a major role in both thrombosis and hemostasis. The expression of TF in monocytes is well documented, but its presence in other blood cells has been disputed, possibly due to methodological variations among different studies. MATERIALS AND METHODS: We studied TF expression on platelets, monocytes, lymphocytes and microparticles (MPs) by flow cytometry (FCM) with five commercially available mouse anti-human TF antibodies (HTF-1, TF9-10H10, CLB/TF-5, VIC7 and VD8). The ability of different TF antibodies to inhibit cell surface TF activity was explored by incubating LPS-stimulated monocytes and MPs derived from LPS-stimulated monocytes (MMPs) with TF antibodies followed by measuring TF activity. RESULTS: HTF-1 detected TF only on LPS-stimulated monocytes, whereas, TF9-10H10 and VD8 detected TF associated with MPs and MMPs in addition to LPS stimulated monocytes. Surprisingly, CLB/TF-5 and VIC7 detected TF on platelets, monocytes even under unstimulated conditions, in addition to MPs and MMPs. CLB/TF-5 also detected TF on unstimulated lymphocytes. Inhibitory studies showed that at a final concentration of 10 µg/mL, HTF-1, CLB/TF-5 and VD8 inhibited monocyte TF activity by 81-84% and MMP TF activity by 92-96%; whereas TF9-10H10 had no inhibitory effect on TF activity in monocytes and MMPs. CONCLUSIONS: Our results suggest non-specific binding by the CLB/TF-5 and VIC7 antibodies in a FCM test system and explain at least some of the reports on TF presence in blood cells, particularly TF associated with platelets and MPs. TF9-10H10 and VD8 are more suitable to detect TF on MPs by FCM.

A wax ester and astaxanthin-rich extract from the marine copepod Calanus finmarchicus attenuates atherogenesis in female apolipoprotein E-deficient mice.

The aim of this study was to investigate the effect of dietary supplementation with an oil extracted from the zooplankton copepod Calanus finmarchicus [calanus oil (CO)] on atherosclerosis in apoE-deficient (apoE(-/-)) mice. Thirty 6-wk-old female apoE(-/-) mice (n = 10/group) were fed: 1) a Western-type, high-fat diet (HFD); 2) HFD supplemented with 1% (wt:wt) CO; or 3) HFD supplemented with 0.88% (wt:wt) corn oil + 0.12% (wt:wt) EPA+DHA ethyl esters (EPA+DHA) for 13 wk. Dietary CO supplementation lowered total aorta atherogenesis by 36.5% compared to the HFD (P < 0.01), whereas the reduction in the lesion prone aortic arch was 34.8% (P < 0.01). The degree of aortic atherogenesis was intermediate in mice fed EPA+DHA compared to those fed HFD and CO. The effect on atherogenesis was paralleled by reduced expression of hepatic genes for the proinflammatory cytokines, Ccl2, Icam1, Il1b, and Nfkb1, in mice fed CO compared to those fed HFD. For mice fed EPA+DHA, gene expression did not differ compared to those fed CO or HFD. Plasma concentrations of total cholesterol, TG, and cytokines did not differ between the groups at the end of the study. However, mice fed CO gained more weight compared to those fed HFD but not compared to those fed EPA+DHA. In conclusion, dietary CO supplementation attenuated atherosclerotic lesion formation in female apoE(-/-) mice and may be an effective and safe dietary intervention to reduce the development of atherosclerosis. However, further studies are warranted to elucidate the underlying physiological and molecular mechanisms.

Shear stress regulates inflammatory and thrombogenic gene transcripts in cultured human endothelial progenitor cells.

Shear stress has an established effect on mature endothelial cells, but less is known about how shear stress regulates endothelial progenitor cells (EPCs). In vitro expanded EPCs isolated from adult human blood represent a novel tool in regenerative vessel therapy. However, in vitro culturing may generate cells with unfavourable properties. The aim of the present study was therefore to assess whether shear stress may influence the inflammatory and thrombotic phenotype of in vitro expanded EPCs. In late outgrowth EPCs, 6 hours of shear stress (6.0 dynes/cm2) significantly reduced the mRNA levels of IL-8, COX2, and tissue factor (TF) compared to static controls. This was associated with a reduced TF activity. In contrast, mRNA expression of NOS3 was significantly increased following 6 and 24 hours of shear stress. In accordance with this, NOS3 protein expression was increased following 24 hours of shear stress. Overall stimulation with the proinflammatory mediator, TNFalpha, for the final 2 hours increased the mRNA expression of IL-6, IL-8, MCP-1, ICAM1, and TF. However exposure to 6 hours of shear stress significantly suppressed the inductory potential of TNFalpha to increase the mRNA levels of IL-6, IL-8, COX2, and TF. Additionally, TNFalpha increased TF activity approximately 10 times, an effect that was also significantly reduced by exposure to 6 and 24 hours of shear stress. The effect of shear on the gene levels of TF and NOS3 were not blocked by the NOS inhibitor L-NAME. These observations suggest that EPCs are capable of functionally responding to shear stress.

Enhanced incorporation of n-3 fatty acids from fish compared with fish oils.

This work was undertaken to study the impact of the source of n-3 FA on their incorporation in serum, on blood lipid composition, and on cellular activation. A clinical trial comprising 71 volunteers, divided into five groups, was performed. Three groups were given 400 g smoked salmon (n = 14), cooked salmon (n = 15), or cooked cod (n = 13) per week for 8 wk. A fourth group was given 15 mL/d of cod liver oil (CLO) (n = 15), and a fifth group served as control (n = 14) without supplementation. The serum content of EPA and DHA before and after intervention revealed a higher rise in EPA and DHA in the cooked salmon group (129% rise in EPA and 45% rise in DHA) as compared with CLO (106 and 25%, respectively) despite an intake of EPA and DHA in the CLO group of 3.0 g/d compared with 1.2 g/d in the cooked salmon group. No significant changes were observed in blood lipids, fibrinogen, fibrinolysis, or lipopolysaccharide (LPS)-induced tissue factor (TF) activity, tumor necrosis factor-alpha (TNFalpha), interleukin-8 (IL-8), leukotriene B4 (LTB4), and thromboxane B2 (TxB2) in whole blood. EPA and DHA were negatively correlated with LPS-induced TNFalpha, IL-8, LTB4, TxB2, and TF in whole blood. In conclusion, fish consumption is more effective in increasing serum EPA and DHA than supplementing the diet with fish oil. Since the n-3 FA are predominantly in TAG in fish as well as CLO, it is suggested that the larger uptake from fish than CLO is due to differences in physiochemical structure of the lipids.

Association of the -159 C --> T polymorphism in the CD14 promoter with variations in serum lipoproteins in healthy subjects.

The CD14-159 C --> T polymorphism, a single nucleotide polymorphism (SNP) at position -159 in the promoter region of the gene encoding the pattern recognition receptor CD14, has been associated with elevated plasma concentrations of soluble CD14, lowered serum immunoglobulin E, increased risk for myocardial infarction, and decreased risk for allergy and asthma. In the present study, the CD14-159 C --> T polymorphism has been investigated in order to determine its frequency and association with proinflammatory variables and lipid profile traits of 117 volunteers. The frequency of the CD14 promoter genotype as determined by polymerase chain reaction amplification-restriction fragment length polymorphism analysis was 35.0% (CC), 44.4% (CT), and 20.5% (TT), and the T allele frequency was 42.7%. Compared with the other genotypes, notably CC homozygotes, TT homozygotes were associated with lower total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B-100 (P < 0.01) concentrations in serum. However, no association was found between the investigated SNP and inflammatory mediators such as fibrinogen, interleukin-6, tumor necrosis factor-alpha, tissue factor, C-reactive protein, plasminogen activator inhibitor-1, leukotriene B4, or thromboxane B2. In conclusion, the CD14-159 C --> T polymorphism may be an important genetic trait, related to the ability of CD14 to bind and transport lipids, such as cholesterol.

Ex-vivo regulation of endotoxin-induced tissue factor in whole blood by eicosanoids.

The influence of several eicosanoids of the lipoxygenase pathway was examined in an ex vivo system of human whole blood subjected to stimulation by lipopolysaccharide (LPS). Exogenously added leukotriene B4 [5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4)] or 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) significantly (P<0.05) enhanced LPS-evoked expression of monocyte tissue factor (TF) activity in a concentration-dependent manner. 15(S)-HETE, on the other hand, exerted such activity only when added at certain concentrations, whereas 5(S)-HETE was devoid of any apparent activity. LPS-induced TF activity was inhibited by the lipoxygenase inhibitors nordihydroguaiaretic acid, CGS 23885 and ZM 230487, by 59, 32 and 88%, respectively. Furthermore, the production of LTB4 in LPS-stimulated whole blood was investigated, in the absence or presence of either tumor necrosis factor alpha (TNFalpha) or phorbol-12-myristate-13-acetate (PMA). LPS alone induced a moderate time-dependent and concentration-dependent release of LTB4, reaching the maximum concentration (1260 +/- 202 pg/ml) within 90 min at 5 ng/ml LPS. The prior and concurrent presence of PMA (5 ng/ml) or TNFalpha (10 ng/ml) further enhanced the LTB4 production approximately twofold (P < 0.05). TNFalpha added alone evoked approximately twice the LTB4 production seen when LPS (2200 +/- 243 versus 1260 +/- 203 pg/ml) was added alone. Considering these results, LPS and TNFalpha emerge as important agonists of LTB4 production in whole blood. LTB4 in turn appears to be of importance for the expression of TF in monocytes, potentially amplifying the thrombogenic potential of these cells.

The effect of soluble beta-1,3-glucan and lipopolysaccharide on cytokine production and coagulation activation in whole blood.

Soluble beta-1,3-glucan has been demonstrated to protect against infection and shock in rats and mice, and clinical studies suggest that administration of soluble glucans to trauma/surgical patients decreases septic complications and improves survival. However, little is known about the precise mechanisms by which glucans influence the state of activation of blood cells, which are responsible for the fulminant cytokine production and the activation of the coagulation system observed in serious gram-negative infection. We studied therefore the effect of an underivatized, soluble yeast beta-1,3-glucan and lipopolysaccharide (LPS), either alone or in combination, on tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 secretion and monocyte tissue factor (TF) expression in human whole blood. As expected, LPS induced the secretion of substantial amounts of all measured parameters, whereas only minor amounts of TNFalpha, IL-6, and IL-10 were induced by beta-glucan itself. However, beta-glucan itself induced the production of significant amounts of IL-8 and TF. Soluble beta-1,3-glucan had a strong synergistic effect on the LPS-induced secretion of IL-8, IL-10, and on monocyte TF activity, but not on TNFalpha and 1L-6 production. On the other hand, soluble beta-glucan strongly primed LPS stimulation of all parameters, including TNFalpha and IL-6. beta-Glucan also induced detectable neutrophil degranulation within 15 min, whereas a response to LPS was first detected after 90 min. In conclusion, soluble beta-1,3-glucan upregulated leukocyte activity, both on its own and in concert with LPS.