Taavo Tenno

Role of Platelet P-Selectin and CD40 Ligand in the Induction of Monocytic Tissue Factor Expression

Activated platelets can express CD40 ligand (CD40L) and trigger inflammatory response and tissue factor (TF) expression in endothelial cells through interaction with CD40. This pathway is also important for T cell–induced monocyte and endothelial cell procoagulant activity. We have studied the potential role of the CD40-CD40L pathway in platelet-induced TF expression in a monocytic cell line and in whole-blood monocytes. In vitamin D3–differentiated U-937 cells, thrombin-stimulated platelets increased TF expression as measured by mRNA quantification, flow cytometry, and procoagulant activity. Maximum antigen expression occurred after 2 hours. Neutralizing anti–P-selectin antibody yielded a 50% suppression of procoagulant activity, whereas antibody to CD40L had no effect. In thrombin receptor activator–stimulated citrated blood, monocytes were up to 77% TF-positive, with peak expression after only 15 minutes. However, no TF mRNA was detectable at that time. Anti–P-selectin antibody reduced TF by 50%, whereas antibody to CD40L gave a 17% reduction. Thus, we conclude that P-selectin exposed on activated platelets induces the expression of TF in both U-937 cells and whole-blood monocytes but by different mechanisms. Platelet CD40L does not display any significant effect on U-937 cells but may be of some importance on whole-blood monocytes. This suggests a possible functional difference between U-937 and monocyte CD40. Another important finding in this study is the rapid appearance of surface TF on monocytes without detectable mRNA formation. This indicates that TF may be stored intracellularly in these cells and can be exposed on the surface independent of de novo protein synthesis.

IL‐10 inhibits LPS‐induced human monocyte tissue factor expression in whole blood

Interleukin 4 (IL‐4), IL‐10 and IL‐13 are all known to modulate several proinflammatory functions in human monocytes. They have also previously been shown to down‐regulate lipopolysaccharide (LPS)‐induced tissue factor (TF) expression in isolated cultured monocytes. In this study we investigated the effect of these three cytokines on the induction of monocytic TF in a whole blood environment at three levels: mRNA quantitation, surface antigen expression and procoagulant activity. We showed that IL‐10 attenuated LPS‐induced monocyte TF expression and activity in whole blood in a concentration‐dependent manner, both when added to the blood prior to LPS and, although to a lesser extent, when added up to 1 h subsequent to LPS challenge. Maximum inhibition occurred at 5 ng/ml of IL‐10 when the cytokine was added before LPS. IL‐4 and IL‐13, however, did not exhibit any inhibitory effect in the whole blood environment, contrary to the reported findings in cell culture experiments. Our results confirm the potential of IL‐10 as an anti‐inflammatory, TF‐preventing drug, whereas the effects of IL‐4 and IL‐13 on monocytes in whole blood seem more complex, and require further investigation.

Genetic Variations in the Tissue Factor Gene Are Associated With Clinical Outcome in Acute Coronary Syndrome and Expression Levels in Human Monocytes

Objective—Tissue factor (TF) has, among other factors, a prominent role in acute coronary syndrome (ACS). Our goal was to investigate whether single nucleotide polymorphisms (SNP) in the TF gene (F3) are associated with plasma TF, risk, and outcome in patients with ACS. Moreover, we wanted to investigate the impact of associated TF SNPs on mRNA production in human monocytes. Methods and Results—In 725 patients with ACS [Fragmin and Fast Revascularization during Instability in Coronary Artery Disease II (FRISC-II) study] and 376 controls, 13 SNPs were genotyped and plasma TF measured. Thereafter, the 5466 A>G and the −1812 C>T were genotyped among all of the FRISC-II participants (n=3143) and assessed concerning clinical outcome. Associated SNPs were genotyped in 92 healthy blood donors for comparison of TF activity and TF mRNA expression. None of the SNPs were associated with patient/control status. The 5466 A>G SNP was associated with cardiovascular death (odds ratio, 1.8; P=0.025). The CG haplotype by −1812 C>T and 5466 A>G was associated with a 3-fold increased risk of death (P<0.001). TF mRNA and basal TF activity was significantly lower among 5466 AG carriers, whereas the increase in monocyte TF activity on lipopolysaccharide stimulation was significantly stronger (P=0.04). Conclusions—The 5466 AG genotype is a novel predictor of cardiovascular death in ACS and may act through a high TF response.

Inhibition of tissue factor surface expression in human peripheral blood monocytes exposed to cytokines.

Interleukin (IL)‐4, IL‐10, IL‐13 and transforming growth factor beta (TGF‐β) are known to regulate several monocyte functions, including inhibition of the synthesis of different cytokines. Using quantitative RT‐PCR and flow cytometry analysis we investigated the effects of these cytokines on bacterial lipopolysaccharide (LPS)‐induced tissue factor (TF) expression in human monocytes. The effects of IL‐4 and IL‐10 on monocyte chemoattractant protein‐1 (MCP‐1)‐ and C‐reactive protein (CRP)‐induced TF expression were also studied. A direct comparison revealed that IL‐4, IL‐10 and IL‐13 all down‐regulated LPS‐induced TF expression in a concentration‐dependent manner without the need for priming. In contrast, TGF‐β required 4 h of priming to inhibit TF expression induced by LPS. IL‐10 was the most powerful inhibitor, causing almost complete inhibition at 5 ng/ml. IL‐4 and IL‐13 exhibited a significantly lower inhibitory capacity even at concentrations of 100 ng/ml. IL‐4 and IL‐10 showed similar concentration‐dependent inhibition of MCP‐1‐ and CRP‐induced TF expression. We also showed that the regulatory effect of the interleukins occurred at the mRNA level. In vivo, these inhibitory cytokines may play an important regulatory role in preventing thrombosis. IL‐10, in particular, may be a possible candidate as a TF‐preventing drug.

Induction of differentiation in U-937 and NB4 cells is associated with inhibition of tissue factor production.

Tissue factor (TF) production is under strict control in mature monocytic cells. However, constitutive expression of TF can be found in myelomonocytic cells and in haematopoietic cells arrested at an early stage of differentiation. In this paper we show that TF expression is down‐regulated during the monocyte/granulocyte differentiation process, using the human monoblastic U‐937 and the acute promyelocytic leukaemia NB4 cell lines as models. Expression of TF mRNA, protein and procoagulant activity (PCA) was constitutively high in untreated cells. Exposure of U‐937 cells to 1 α,25‐dihydroxycholecalciferol (VitD3) and all‐trans retinoic acid (ATRA) resulted in down‐regulation of TF expression and PCA. In NB4 cells induction by ATRA, but not VitD3, resulted in the down‐regulation of TF expression and PCA. Consistent with this, induction of terminal differentiation, as confirmed by the expression of differentiation associated antigens and cell cycle arrest, was inversely correlated to TF expression in U‐937 and NB4 cells. Moreover, terminally differentiated U‐937 cells retained the capacity to respond to inflammatory mediators, i.e. lipopolysaccharide and interferon‐γ, by a rapid increase in TF expression. In conclusion, we show that not only ATRA but also VitD3 is a potent suppressor of monocytic TF expression and thus might have potential clinical use for the treatment of coagulopathies.

Monocytes, but not macrophages, produce the eosinophil cationic protein

The eosinophil cationic protein (ECP) is a cytotoxic protein with ribonuclease activity, produced and stored in bone marrow eosinophil myelocytes. Mature circulating eosinophils contain about 10 pg ECP per cell. The aim of this study was to investigate the possibility that monocytes produce and store ECP. By results from flow cytometry and specific protein measurement it is shown that human monocytes contain ECP (monocytes about 10 fg ECP per cell). RT‐PCR analysis indicated the presence of mRNA coding for ECP in blood monocytes but not in alveolar macrophages. Furthermore, mRNA coding for ECP and low amounts of the protein were found in three myeloid cell lines representing different stages of monocytic differentiation. Differentiation of U‐937 cells to macrophages induced lowered transcription of the ECP gene and reduced protein production. Immunohistochemical staining of lung tissue indicated that lung macrophages do not contain ECP. It is concluded that ECP is produced to a low extent by human monocytes and that the production is shut down during macrophage differentiation. This might indicate an alternative transcriptional regulation of the ECP gene in the monocytic lineage compared to the eosinophil lineage.

The role of RAR and RXR activation in retinoid-induced tissue factor suppression

Excessive expression of tissue factor (TF) is a common finding in leukaemic cells and may contribute to thrombotic complications in patients. Retinoic acid has been shown to induce differentiation and reduce TF expression in acute promyelocytic leukaemia (APL) cells in vitro, and to induce remission in APL patients. Treatment of the APL cell line NB4 with the specific retinoic acid receptor-α (RARα) agonists Ro40–6055 or TTNPB resulted in down-regulation of TF expression and in induction of differentiation. The activation of RARβ, RARγ or retinoid X receptor (RXR) did not suppress the constitutive TF expression in NB4 cells. Moreover, the RARα antagonist Ro41-5253 blocked the retinoid-induced down-regulation of TF. In contrast, in the monoblastic U-937 cell line only a partial suppression of TF antigen expression and activity was observed by treatment with the RAR agonist TTNPB or the RXR agonist SR11237 alone. However, the combination of TTNPB and SR11237 resulted in a pronounced down-regulation of TF expression and induction of differentiation in U-937 cells. We show for the first time that the activation of both subunits of the RARα-RXR transcriptional complex is needed for TF suppression in U-937 cells, whereas in NB4 cells RARα activation alone is sufficient. Thus, distinct molecular mechanisms for TF suppression seem to be operating in leukaemic cell lines of different origin.

TISSUE FACTOR EXPRESSION IN HUMAN MONOCYTIC CELL LINES

Tissue factor (TF) is a main initiator of the coagulation protease cascade. Control of the expression of this protein in monocytes is essential, since these cells are the only circulating blood cells responsible for TF expression. In this report we have used two human cell lines, arrested at different stages of monocytic differentiation, to study TF expression. The monoblastic cell line U-937 had a constitutive expression of TF surface protein and low TF mRNA levels detected by immunofluorescence or quantitative reverse transcriptase polymerase chain reaction respectively. The phorbol-12-myristate-13-acetate (PMA) was a potent enhancer of TF expression in U-937. Lipopolysaccharide (LPS) and tumor necrosis factor (TNF) had no effect on TF expression in U-937. The Mono Mac 6 cell line, with phenotypic features similar to that of mature monocytes, expressed lower basal levels of TF mRNA and surface TF antigen. However, in Mono Mac 6 cells TF expression was induced in response to LPS and TNF. These results indicate differences in basal and induced TF expression between U-937 and Mono Mac 6 cell lines.