Monocytes and plasma tissue factor levels in normal individuals and patients with deep venous thrombosis of the lower limbs: potential diagnostic tools?

INTRODUCTION: Tissue factor (TF) is the main physiological initiator of blood coagulation; it is membrane-bound on monocytes (mTF) and free in plasma (pTF). Abnormal expression of TF by monocytes has been implicated in various diseases. We therefore quantified monocytes expressing TF and pTF levels in patients with lower-limb deep venous thrombosis (DVT). MATERIALS AND METHODS: DVT was confirmed by Duplex Scan. Blood mTF levels under resting condition (baseline), after incubation without (unstimulated) and with (stimulated) lipopolysaccharide (LPS), and total mTF levels were determined by flow cytometry using two analytical methods (Histogram and Quadrant-Statistics). Plasma TF levels were measured using an enzyme-linked immunoabsorbent assay (ELISA). Results were compared with age-matched controls. RESULTS: Histogram analysis in patients with DVT showed significantly elevated mTF levels for baseline, unstimulated and total mTF over controls. For Quadrant-Statistics, DVT patients also showed significantly raised baseline, unstimulated, stimulated and total mTF. Similarly, pTF levels were significantly raised in subjects with DVT compared to controls. Baseline mTF levels correlated with pTF levels by Histogram and Quadrant-Statistics analysis. Using the relative operating characteristic (ROC) curve, baseline mTF and pTF assays displayed sensitivity and specificity in detecting DVT. Quadrant-Statistics baseline mTF and pTF gave the best discrimination. CONCLUSIONS: The TF assays used in this study showed acceptable sensitivity and specificity and are cost-effective and practical. Therefore, they should be considered in patients with, or at risk of, DVT.     

Primary rat monocytes migrate through a BCEC-monolayer and express microglia-markers at the basolateral side

Monocytes are pluripotent cells of the immune system, circulate in the blood and cross the blood-brain barrier continuously through life. The aim of this study was to explore if primary rat monocytes can adhere and transmigrate at a monolayer of brain capillary endothelial cells (BCEC) and if the monocytes undergo differentiation toward a microglial phenotype at the basolateral side. Monocytes and as a control primary microglia were immunohistochemically stained with markers for CD68 (clone ED-1), CD11b (clone OX-42) or CD11c (clone 8A2). The primary rat monocytes (100,000 cells added) adhered at the BCEC-monolayer (approx. 1200 cells/well) within 30 min and migrated to the basolateral side within 18 h (approx. 40,000 cells/well). The transmigrated monocytes partly differentiated and expressed microglia-markers at the basolateral side. Tumor necrosis factor-alpha as well as conditioned medium derived from BCEC stimulated the differentiation of monocytes in culture. In conclusion, monocytes adhere and migrate through a BCEC-monolayer and express microglia-markers at the basolateral side.     

TLR8‐mediated activation of human monocytes inhibits TL1A expression

TLR play important roles in inflammation and innate immune response to pathogens. TLR8 recognizes ssRNA and induces NF‐κB via MyD88 signaling. TL1A is a member of the TNF superfamily that markedly enhances IFN‐γ production by IL‐12/IL‐18‐stimulated peripheral and mucosal CD4⁺ T cells. TL1A expression is increased in the mucosa of patients with inflammatory bowel disease and is considered a key mediator of Crohn's disease (CD). We have previously shown that TL1A is strongly induced by immune complexes (IC) but not TLR ligands in antigen‐presenting cells. However, a potential interaction between these pro‐inflammatory signaling pathways has not been investigated. IC‐induced TL1A expression of monocytes was potently inhibited by a TLR8 or TLR7/8 ligand (R848) in a dose‐dependent manner. Furthermore, when co‐cultured with CD4⁺ T cells, TLR8 ligands inhibited TL1A production, resulting in almost complete inhibition of IFN‐γ production by the CD4⁺ T cells. Furthermore, we demonstrate that IFN‐α is not required for this suppressive effect by TLR8 signaling. Our data demonstrate for the first time a direct interaction between TLR and TL1A signaling pathways. TLR8 activation may be an important, novel pathway for targeted treatment of Th1‐mediated diseases, such as CD.     

Depletion of macrophages in mice results in higher dengue virus titers and highlights the role of macrophages for virus control

Monocytes and macrophages are target cells for dengue infection. Besides their potential role for virus replication, activated monocytes/macrophages produce cytokines that may be critical for dengue pathology. To study the in vivo role of monocytes and macrophages for virus replication, we depleted monocytes and macrophages in IFN‐αβγR knockout mice with clodronate liposomes before dengue infection. Although less virus was first recovered in the draining LN in the absence of macrophages, monocyte/macrophage depletion eventually resulted in a ten‐fold higher systemic viral titer. A massive infiltration of CD11b⁺CD11c^lowLy6C^low monocytes into infected organs was observed in parallel with increasing virus titers before viremia was controlled. Depletion of monocytes in the blood before or after local infection had no impact on virus titers, suggesting that monocytes are not required as “virus‐shuttles”. Our data provide evidence that systemic viremia is established independently of tissue macrophages present at the site of infection and blood monocytes. Instead, we demonstrate the importance of monocytes/macrophages for the control of dengue virus.     

定量PCR检测热性惊厥患儿外周血单个核细胞ICAM-1／LFA-1 mRNA表达水平

目的探讨细胞间黏附分子-1（ICAM-1）及其配体淋巴细胞功能相关抗原-1（LFA-1）对热性惊厥（FS）患儿的神经免疫调节作用。方法40例FS患几分为单纯性FS（SFS）组20例和复杂性FS（CFS）组20例;对照组19例。分别采用实时荧光定量PCR、RT-PCR方法检测外周血单个核细胞（PBMC）ICAM-1／LFA-1 mRNA的表达水平。结果SFS组患儿ICAM—1 mRNA水平为（0．63±0．39）基因copy数,明显高于对照组（0．46±0．20）和CFS组患儿（0．41±0．20）基因copy数（P〈0．05）;CFS组ICAM—1 mRNA水平与对照组儿童比较有下调趋势,但差异无统计学意义（P〉0．05）。SFS组LFA—1 mRNA表达水平为（0．73±0．11）灰度值比。明显高于对照组（0．60±0．15）和CFS组（0．54±0．17）灰度值比（P〈0．05）;CFS组患儿LFA-1 mRNA水平与对照组儿童比较亦有下调趋势,但差异无统计学意义（P〉0．05）。结论PBMC ICAM-1／LFA-1基因表达水平在SFS和CFS是不同的,ICAM-1／IFA-1 mRNA表达增高所介导的炎性免疫病理损害在SFS患儿发病机制中有一定的作用,而CFS患儿ICAM-1／LFA-1 mRNA表达反而下调可能有一定的神经保护作用。     

PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes

The objective of the present study was to investigate the toxicity of silver nanoparticles (Ag NPs) in vitro. Silver ions (Ag+) have been used in medical treatments for decades whereas Ag NPs have been used in a variety of consumer products within recent years. This study was undertaken to compare the effect of well characterized, PVP-coated Ag NPs (69 nm ± 3 nm) and Ag+ in a human monocytic cell line (THP-1). Characterization of the Ag NPs was conducted in both stock suspension and cell media with or without serum and antibiotics. By using the flowcytometric annexin V/propidium iodide (PI) assay, both Ag NPs and Ag+ were shown to induce apoptosis and necrosis in THP-1 cells depending on dose and exposure time. Furthermore, the presence of apoptosis could be confirmed by the TUNEL method. A number of studies have implicated the production of reactive oxygen species (ROS) in cytotoxicity mediated by NPs. We used the fluorogenic probe, 2′,7′-dichlorofluorescein to assess the levels of intracellular ROS during exposure to Ag NPs and Ag+. A drastic increase in ROS levels could be detected after 6–24 h suggesting that oxidative stress is an important mediator of cytotoxicity caused by Ag NPs and Ag+.     

Reduced expansion rate of abdominal aortic aneurysms in patients with diabetes may be related to aberrant monocyte-matrix interactions

Aims: Diabetes increases the risk of atherothrombosis, but reducesthe risk of abdominal aortic aneurysm (AAA). The reason forthis difference is unknown. We examined the role of diabetesand glycation on AAA expansion and extracellular matrix–monocyteinteractions. Methods and results: We followed 198 patients (20 with diabetes) who had 30–45mm AAAs with yearly aortic ultrasound for 3 years. Diabeteswas independently associated with reduced AAA growth (β= –0.17, P = 0.01; OR for expansion above median 0.18,95% confidence interval 0.06–0.57). In vitro incubationof resting human monocytes with glycated bovine serum albuminor monomeric type I collagen increased matrix metalloproteinase(MMP) secretion. In contrast, exposure of activated monocytesto glycated type I collagen lattices induced a marked reductionin MMP and interleukin-6 secretion. This de-activating effectwas also demonstrated in cross-linked non-glycated collagenlattices, healthy decellularized aortic media, and decellularizedaortic media from diabetes patients with atherosclerosis. Incontrast, decellularized aortic media from patients with atherosclerosis,but no diabetes, induced increased MMP secretion. Conclusion: These findings confirm that the progression of AAA is slowerin patients with diabetes and suggest a mechanism by which theaortic media may be protected from degradation in these individuals.     

白细胞介素6诱导的组织因子表达在慢性排斥反应发病机制中的作用

目的研究白细胞介素6（IL-6）对人外周血单核细胞（PBMCs）中组织因子（TF）的表达及活性的诱导作用。方法分别用人重组白细胞介素6（rhIL-6）100ng／L和rhIL-6100ng／L＋rhIL-6单克隆抗体（MoAb）10μg／L培养刺激PBMCs24h，将未经刺激的PBMCs作为对照组，观察各组细胞中TF抗原含量、mRNA的表达及TF活性的变化。结果经rhIL-6培养刺激24h后，PBMCs的TF抗原含量、mRNA的表达及TF活性均较对照组和rhIL-6＋rhIL-6 MoAb组显著增加（P〈0．01）。结论rhIL-6可诱导PBMCs的TF抗原表达和活性，并使mRNA的表达增加，rhIL-6 MoAb能有效抑制rhIL-6所致的PBMCs的TF表达及活性，提示炎症因子与凝血机制的启动存在内在联系，并在慢性排斥反应中起重要作用。     

Changes in the choroid plexus,responses by intrinsic epiplexus cells and recruitment from monocytes after experimental head acceleration injury in the non-human primate

Summary We have examined, by scanning and transmission electron microscopy, morphological changes in the choroid plexus of the lateral ventricles of the non-human primate brain after lateral head acceleration. We demonstrate passage of plasma and blood cells either through tears in blood vessels and the choroidal epithelium, or through the cells of the choroidal epithelium, 20 min after injury, together with morphological changes in that epithelium. At 3 and 4 h small cells with a reniform nucleus accumulate in the connective tissue core of the choroid plexus. We suggest that these are monocytes. At 6 and 12 h cells can be seen in enlarged intercellular spaces within the choroidal epithelium. These cells possess surface ruffles and we suggest that they are monocytes differentiating into macrophages and epiplexus cells. Further evidence for transepithelial migration of monocytes/macrophages is obtained at 7 days. However, at 28 days all blood has been removed from the surface of the choroid plexus and epiplexus cells possess an appearance typical of that in uninjured animals. The possible sources of epiplexus cells are discussed with reference to studies of responses after brain insult and of development. We have obtained no evidence in support of emperipolesis by monocytes through the choroidal epithelium. We suggest that monocytes/macrophages migrate, via an intercellular route, to differentiate into epiplexus cells, thus providing additional numbers of epiplexus cells after head injury.     

Intercellular adhesion molecule 1 on monocytes mediates adhesion as well as trans-endothelial migration and can be downregulated using antisense oligonucleotides

The intercellular adhesion molecule 1 (ICAM-1) on endothelial cells is involved in the recruitment of leukocytes to inflammatory sites. In contrast to ICAM-1 expression on endothelial cells, little is known about its function in leukocytes in inflammation. Using ICAM-1-directed anti-sense oligodeoxyribonucleotides (ODNs), we examined the role of ICAM-1 expression on monocytes and lymphocytes for adhesion and trans-endothelial migration. As determined by flow cytometry, a downregulation of the ICAM-1 expression of 50 % was observed on peripheral blood mononuclear cells (PBMCs) after their transfection with anti-sense ODNs using cationic lipids. The decrease in the level of ICAM-1 expression in PBMCs was associated with a 36% inhibition of adhesion to interleukin-1β-stimulated endothelial cells and a 40% reduction of trans-endothelial migration. Gating on particular subsets of the PBMC, the downregulation of ICAM-1 and the functional effects could be ascribed to monocytes, while no significant inhibition was found for lymphocytes. This could be explained by differences in cellular ODN uptake. Since the ligands of ICAM-1 are not expressed on endothelial cells, the results suggest a homotypic interaction among monocytes. In conclusion, in addition to ICAM-1 expression on endothelial cells, ICAM-1 expression on monocytes mediates adhesion and trans-endothelial migration. This might be relevant for the clinical use of ICAM-1-directed anti-sense ODNs for the treatment of inflammatory diseases, because monocytes appear to be suitable target cells in which to achieve anti-inflammatory effects. Received: 25 June 1999 / Accepted: 2 December 1999