The Blood Monocyte - TNF - Endothelial Axis in Activation of Endothelial Tissue Factor in the Transgenic Sickle Mouse: Possible Relevance of Etanercept to Sickle Coagulopathy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1048-1048
Author(s):  
Anna Solovey ◽  
Liming Milbauer Chang ◽  
Fuad Abdulla ◽  
Rahn Kollander ◽  
Paul H Marker ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Pulmonary Veins ◽  
Ambient Air ◽  
Blood Monocytes ◽  
Extended Treatment ◽  
Pre Treatment ◽  
Endothelial Tissue

Abstract Abstract 1048 Peripheral blood monocytes can be pathogenic participants in vascular disease, and they are abnormally activated in sickle patients. As shown earlier, sickle mice have abnormally increased expression of endothelial tissue factor (eTF) in pulmonary veins (PV), expressed as % of PV positive for eTF. For HbS-BERK (eTF 36%, vs 9% for HbA-BERK controls) and S+SAntilles (eTF 28%, vs 7% for C57BL6 controls), abnormal eTF expression is chronic. For NY1DD, however, eTF switches from 7% at ambient air to 23% after exposure to hypoxia/reoxygenation (post-H/R). Having now examined roles of transcription factors Egr-1 and NFkB(p50), we find that H/R triggered expression of eTF in post-H/R NY1DD is: [a] dependent upon both Egr-1 and NFkB(p50) within peripheral blood mononuclear cells (PBMC), and dependent upon Egr-1 butnot NFkB(p50) in vessel wall endothelium. To examine the roles of PBMC and their product TNF in activating eTF expression we used three strategies. [A] First, transfusion (Tx) of PBMC from donor mice (DM) induced eTF in recipient mice (RM) as follows. At-air NY1DD RM exhibited eTF of 7% from Tx of at-air NY1DD DM PBMC, and eTF of 19% from Tx of post-H/R NY1DD DM PBMC. C57BL6 RM exhibited eTF of 10% from Tx of C57BL6 DM PBMC, 31% from Tx of S+SAntilles DM PBMC, and only 15% from Tx of PBMC from S+SAntilles DM also having NFkB(p50)−/−. HbA-BERK RM exhibited eTF of 9% from Tx of HbA-BERK DM PBMC, and 36% from Tx of HbS-BERK DM PBMC. [B] Second, we examined effect of TNF+LT blocker etanercept on PBMC in transfusion experiments. As expected, HbA-BERK RM exhibited eTF 36% from Tx of HbS-BERK PBMC obtained from etanercept-treated (3 mg/kg × 2) DM, and 38% from Tx of HbS-BERK PBMC obtained from DM treated with inactive control TNF-R-scrambled-peptide. On the other hand, HbA-BERK RM pre-treated with etanercept (same dose) vs control TNF-R-scrambled-peptide exhibited eTF of 11% and 33%, respectively from Tx of HbS-BERK DM PBMC. [C] Third, direct TNF blocking pre-treatment of NY1DD mice prevented the eTF increase from subsequent H/R (to 23% for the no pre-treatment controls): eTF of 5% (P=.000013) for pre-treatment with high-etanercept (10 mg/kg × 2), 12% (P=.00059) for pre-treatment with low-etanercept (3 mg/kg × 2), 19% (P=.053) for pre-treatment with IL1-blocker anakinra (10 mg/kg ×2), and 7% (P=.0016) for pretreatment withTNF-specific blocker infliximab (10 mg/kg ×2). For HbS-BERK mice having pre-existing eTF of 36%, extended treatment with etanercept (3 mg/kg, twice weekly × 3) reduced eTF to 27% (P=.026) while those receiving scrambled-peptide remained at 34%. Higher or longer etanercept may be more effective. Other murine treatments have shown us reversal of pre-existing eTF requires extended treatment. Since regulation of TF expression in endothelial cells and blood monocytes is similar, it is likely that these eTF effects would be paralleled by similar effects on monocyte TF expression. Thus, these results suggest that TNF blocking strategies could be helpful in mitigating effects of coagulation activation in the sickle context. Disclosures: No relevant conflicts of interest to declare.

scholarly journals C-reactive protein induces human peripheral blood monocytes to synthesize tissue factor

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 513-520 ◽  
Author(s):  
J Cermak ◽  
NS Key ◽  
RR Bach ◽  
J Balla ◽  
HS Jacob ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
De Novo ◽  
Human Peripheral Blood ◽  
Umbilical Vein ◽  
Blood Monocytes ◽  
C Reactive Protein ◽  
Dot Blot ◽  
Reactive Protein

The acute inflammatory response is frequently accompanied by serious thrombotic events. We show that C-reactive protein (CRP), an acute- phase reactant that markedly increases its serum concentration in response to inflammatory stimuli, induced monocytes to express tissue factor (TF), a potent procoagulant. Purified human CRP in concentrations commonly achieved in vivo during inflammation (10 to 100 micrograms/mL) induced a 75-fold increase in TF procoagulant activity (PCA) of human peripheral blood mononuclear cells (PBM), with a parallel increase in TF antigen levels. CRP-induced PCA was completely blocked by a monoclonal antibody against human TF but not by irrelevant murine IgG. Dot blot analysis showed a significant increase of TF mRNA after 4 hours of incubation with CRP, followed by a peak of PCA within 6 and 8 hours. Actinomycin D and cycloheximide blocked CRP-stimulated PCA, suggesting that de novo TF protein synthesis was required. Endotoxin (LPS) contamination of CRP was excluded as the mediator of TF synthesis because: (1) CRP was Limulus assay negative; (2) induction of TF PCA by CRP was not blocked by Polymyxin B, in contrast to LPS- induced PCA; (3) antihuman CRP IgG inhibited CRP-induced PCA, but not LPS-induced PCA; (4) CRP was able to stimulate TF production in LPS- pretreated PBM refractory to additional LPS stimulation; and, (5) unlike LPS, CRP was incapable of inducing TF in human umbilical vein endothelial cells. We suggest that CRP-mediated TF production in monocytes may contribute to the development of disseminated intravascular coagulation and thrombosis in inflammatory states.

scholarly journals C-reactive protein induces human peripheral blood monocytes to synthesize tissue factor

Blood ◽  
1993 ◽  
Vol 82 (2) ◽  
pp. 513-520 ◽  
Author(s):  
J Cermak ◽  
NS Key ◽  
RR Bach ◽  
J Balla ◽  
HS Jacob ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
De Novo ◽  
Human Peripheral Blood ◽  
Umbilical Vein ◽  
Blood Monocytes ◽  
C Reactive Protein ◽  
Dot Blot ◽  
Reactive Protein

Abstract The acute inflammatory response is frequently accompanied by serious thrombotic events. We show that C-reactive protein (CRP), an acute- phase reactant that markedly increases its serum concentration in response to inflammatory stimuli, induced monocytes to express tissue factor (TF), a potent procoagulant. Purified human CRP in concentrations commonly achieved in vivo during inflammation (10 to 100 micrograms/mL) induced a 75-fold increase in TF procoagulant activity (PCA) of human peripheral blood mononuclear cells (PBM), with a parallel increase in TF antigen levels. CRP-induced PCA was completely blocked by a monoclonal antibody against human TF but not by irrelevant murine IgG. Dot blot analysis showed a significant increase of TF mRNA after 4 hours of incubation with CRP, followed by a peak of PCA within 6 and 8 hours. Actinomycin D and cycloheximide blocked CRP-stimulated PCA, suggesting that de novo TF protein synthesis was required. Endotoxin (LPS) contamination of CRP was excluded as the mediator of TF synthesis because: (1) CRP was Limulus assay negative; (2) induction of TF PCA by CRP was not blocked by Polymyxin B, in contrast to LPS- induced PCA; (3) antihuman CRP IgG inhibited CRP-induced PCA, but not LPS-induced PCA; (4) CRP was able to stimulate TF production in LPS- pretreated PBM refractory to additional LPS stimulation; and, (5) unlike LPS, CRP was incapable of inducing TF in human umbilical vein endothelial cells. We suggest that CRP-mediated TF production in monocytes may contribute to the development of disseminated intravascular coagulation and thrombosis in inflammatory states.

Endothelial cell expression of tissue factor in sickle mice is augmented by hypoxia/reoxygenation and inhibited by lovastatin

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 840-846 ◽  
Author(s):  
Anna Solovey ◽  
Rahn Kollander ◽  
Arun Shet ◽  
Liming C. Milbauer ◽  
Stephana Choong ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Pulmonary Veins ◽  
Ambient Air ◽  
Tissue Expression ◽  
Circulating Endothelial Cells ◽  
Blood Monocytes ◽  
Tissue Sections ◽  
In The Wild ◽  
Normal Human ◽  
Cell Expression

AbstractAbnormal tissue factor (TF) expression has been demonstrated on blood monocytes and circulating endothelial cells in humans with sickle cell anemia. We have now studied sickle transgenic mice to help define the biology of endothelial TF expression in sickle disease. Using immunostaining of tissue sections, we find that this is confined almost exclusively to the pulmonary veins. About 15% and 13% of these exhibit TF-positive endothelium in the wild-type normal mouse and the normal human hemoglobin (HbA)–expressing control transgenic mouse, respectively. The mild sickle mouse is indistinguishable from normal (∼ 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (∼ 29% and ∼ 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF expression phenotype to that of the severe sickle mouse (∼ 36% positive). Pretreatment with lovastatin eliminated excessive expression of TF in the posthypoxic mild sickle mouse (∼ 16% positive) and in the more severe mouse at ambient air (∼ 21% positive). In addition to identifying tissue expression of endothelial TF in the sickle lung, these studies implicate reperfusion injury physiology in its expression and suggest a rationale for use of statins in sickle disease.

scholarly journals Glucocerebrosidase Activity is Reduced in Cryopreserved Parkinson’s Disease Patient Monocytes and Inversely Correlates with Motor Severity

2021 ◽  
pp. 1-9
Author(s):  
Laura P. Hughes ◽  
Marilia M.M. Pereira ◽  
Deborah A. Hammond ◽  
John B. Kwok ◽  
Glenda M. Halliday ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Rating Scale ◽  
Disease Patient ◽  
Motor Symptom ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Classical Monocytes

Background: Reduced activity of lysosomal glucocerebrosidase is found in brain tissue from Parkinson’s disease patients. Glucocerebrosidase is also highly expressed in peripheral blood monocytes where its activity is decreased in Parkinson’s disease patients, even in the absence of GBA mutation. Objective: To measure glucocerebrosidase activity in cryopreserved peripheral blood monocytes from 30 Parkinson’s disease patients and 30 matched controls and identify any clinical correlation with disease severity. Methods: Flow cytometry was used to measure lysosomal glucocerebrosidase activity in total, classical, intermediate, and non-classical monocytes. All participants underwent neurological examination and motor severity was assessed by the Movement Disorders Society Unified Parkinson’s Disease Rating Scale. Results: Glucocerebrosidase activity was significantly reduced in the total and classical monocyte populations from the Parkinson’s disease patients compared to controls. GCase activity in classical monocytes was inversely correlated to motor symptom severity. Conclusion: Significant differences in monocyte glucocerebrosidase activity can be detected in Parkinson’s disease patients using cryopreserved mononuclear cells and monocyte GCase activity correlated with motor features of disease. Being able to use cryopreserved cells will facilitate the larger multi-site trials needed to validate monocyte GCase activity as a Parkinson’s disease biomarker.

scholarly journals Evaluation of the phagocytic activity of peripheral blood monocytes of patients with Jorge Lobo's disease

2004 ◽  
Vol 37 (2) ◽  
pp. 165-168 ◽  
Author(s):  
Fátima Regina Vilani-Moreno ◽  
Luciana Moreira Silva ◽  
Diltor Vladimir Araújo Opromolla
Keyword(s):  
Incubation Time ◽  
Peripheral Blood ◽  
Phagocytic Activity ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Significant Difference ◽  
Host Parasite ◽  
And Control

Studies on host-parasite interaction in Jorge Lobo's disease are scarce, with no report in the literature on the phagocytosis of Lacazia loboi by phagocytic mononuclear cells. Thus, the objective of the present study was to assess the phagocytic activity of blood monocytes in the presence of L. loboi in patients with the disease and in healthy subjects (controls) over 3 and 24 hours of incubation. Statistical analyses of the results showed no significant difference in percent phagocytosis of the fungus between patient and control monocytes. With respect to incubation time, however, there was a significant difference, in that percent phagocytosis was higher at 3 hours than at 24 hours (p <0.01). These results suggest that monocytes from patients with the mycosis are able to phagocyte the fungus, as also observed in control individuals.

scholarly journals Peroxynitrite reduces endotoxin-induced tissue factor expression in human peripheral blood monocytes

Critical Care ◽  
10.1186/cc29 ◽  
1997 ◽  
Vol 1 (Suppl 1) ◽  
pp. P023
Author(s):  
M Gerlach ◽  
D Keh ◽  
S Spielmann ◽  
T Kerner ◽  
R Peter ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Tissue Factor Expression ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Factor Expression

Therapeutic Inhibition of Endothelial Cell Tissue Factor Expression In Vivo by Nitric Oxide and Arginine in Sickle Transgenic Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 210-210
Author(s):  
Robert P. Hebbel ◽  
Rahn Kollander ◽  
Anna Solovey ◽  
Stephana Choong ◽  
Robert J. Kelm
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Pulmonary Veins ◽  
Dietary Supplementation ◽  
Ambient Air ◽  
Circulating Endothelial Cells ◽  
Coagulation System ◽  
Mild Phenotype

Abstract Sickle cell anemia is accompanied by biochemical activation of the coagulation system and by clinical thromboses. Therefore, we have focused on the expression of tissue factor (TF), the trigger of the coagulation system, in this disease. Previous studies from our laboratories described abnormal TF expression by monocytes and by circulating endothelial cells in human sickle blood. More recently, we reported (Blood104:840, 2004) that severe-phenotype (BERK) sickle mice abnormally express TF on pulmonary vein endothelium; mild-phenotype (NY1DD) sickle mice are normal in this regard but assume the abnormal phenotype after exposure to hypoxia (3 hr at 8% O2) followed by reoxygenation for 18 hr at ambient air (H/R). Monitoring the % of pulmonary veins positive for TF on triple stained tissue sections (for nuclei, for murine TF, for murine CD31 to identify endothelial cells), we have now evaluated the role of NO in TF expression, employing these two models. In the NY1DD H/R model, NO inhalation (34 ppm) administered during the entire reoxygenation period led to 68±18% reduction (P<.001) of TF expression. Consistent with this, dietary supplementation with arginine (substrate for nitric oxide synthase [NOS]) prior to H/R led to 93±25% reduction (P<.001) of the H/R induced TF response. Conversely, dietary supplementation with L-NAME (inhibitor of NOS) converted the NY1DD mouse to a TF positive phenotype (P=.047) without even requiring H/R. Interestingly, inhalation of NO was not protective in the NY1DD H/R model if given only during the hypoxia period itself or only during the last 3 hours of the reoxygenation period, but it was protective if given only during the first 3 hours of the reoxgygenation period (68±24% reduction; P=.0008). Thus, the timing of clinical intervention is probably critical. In the BERK model at ambient air, treatment with dietary arginine for 2 weeks diminished TF expression by 60±33% (P=.04), an effect that was countervailed by concurrent administration of L-NAME. Data will soon be available on eNOS over-expressing sickle mice. Also in BERK mice, L-NAME largely prevented the TF inhibiting therapeutic benefit of lovastatin, suggesting that lovastatin’s beneficial effect on TF is mediated via NO biology. These results suggest that NO biology determines endothelial TF expression in vivo in sickle mice. We note that NO is reported to suppress endothelial TF expression in vitro. We find that sickle mice have elevated plasma free Hb levels (increased by 40% in NY1DD mice and 57% in BERK mice), a risk factor for diminished NO bioavailability. On the other hand, H/R in NY1DD mice is not associated with a further increase in free plasma Hb. So it seems that the NO biology of sickle mice is not simply explained by plasma Hb. NO biology seems to exert a major role in regulating endothelial TF expression in the sickle mouse. Long-term NO modulating therapy, such as dietary arginine or lovastatin, may be beneficial in terms of the coagulopathy and thrombosis risk of sickle patients and should be tested with this in mind.

Characterization of Receptors Involved in Heparin Antibody Complex Mediated Induction of Tissue Factor Expression in Monocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 224-224 ◽  
Author(s):  
Sam Glover ◽  
Nigel S. Key ◽  
Gowthami M Arepally ◽  
Nigel Mackman ◽  
Raj S. Kasthuri
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Drug Induced ◽  
Peripheral Blood Mononuclear ◽  
Platelet Factor ◽  
Antibody Complex

Abstract Abstract 224 Introduction: Heparin-induced thrombocytopenia (HIT) is a major cause of drug-induced thrombocytopenia and occurs in 1–5% of individuals exposed to heparin. Paradoxically, 30–50% of individuals with HIT develop thrombosis. The mechanism of thrombosis in HIT is poorly understood. We recently reported that HIT antibody complexes induce tissue factor (TF) expression in monocytes and result in the release of TF-positive microparticles (MPs). The mechanism by which HIT antibody complexes induce monocyte TF has not been established. The objective of this study is to characterize the receptors involved in HIT antibody complex mediated induction of TF expression in monocytes. As HIT antibody complex mediated activation of platelets is dependent on the FcgRIIA receptor, we evaluated the role of the FcgRII receptor in the induction of monocyte TF by HIT antibody complexes. We also evaluated the role of toll like receptor-4 (TLR4) and the platelet factor 4 (PF4) chemokine receptor CXCR3 in this process. Methods: The combination of heparin, PF4 and the murine monoclonal PF4/heparin-specific antibody KKO has been shown to cause activation of platelets and monocytes, and mimic HIT in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were pre-incubated for 30 min at 37°C with an inhibitory antibody to the FcgRII receptor (IV.3); anti-CXCR2, 3, or 4 antibodies; anti-TLR4 antibody; or mouse-IgG (mIgG) control. Following pre-incubation with antibodies for 30 minutes, heparin (1U/mL), PF4 (10μg/mL), and KKO (100μg/mL) – together referred to as the HIT antibody complex – were added. Heat-aggregated mIgG and LPS were used as positive controls for the FcgRII and TLR4 receptors, respectively. Following a 6-hour incubation, PBMCs were pelleted by centrifugation and MPs were isolated from the supernatant. The procoagulant activity (PCA) of PBMCs and MPs was measured using clotting assays performed in the presence of the anti-TF antibody HTF-1 or control antibody. TF dependent PCA was calculated by reference to a standard curve generated using relipidated recombinant TF. Results: Incubation of PBMCs with heat aggregated mIgG for 6 hours resulted in significant induction of cellular TF (345 +/− 36 pg/106 cells) which was blocked by 30 min pre-incubation with the antibody IV.3 (146 +/− 17 pg/106 cells, N=3, p<.003). However, pre-incubation with IV.3 had no significant effect on TF induction (140 +/− 5 pg/106 cells) associated with the HIT antibody complex when compared to control mIgG (110 +/− 18 pg/106 cells, N=3, p<0.11). PBMCs incubated with HIT antibody complexes in the presence of a TLR-4 antibody showed less TF activity (52 +/− 4 pg/106 cells) compared to control mIgG (80 +/− 10 pg/106 cells N=3, p<0.025). A similar, partial inhibition of TF activity was also observed in PBMCs incubated with LPS in the presence of an anti-TLR4 antibody (121 +/− 3 pg/106) compared with a control antibody (89 +/− 2 pg/106, N=3, p<.0013). Experiments with a more effective inhibitor of TLR4 are in progress. PBMCs incubated with the HIT antibody complexes in the presence of an anti-CXCR3 antibody showed less TF activity (36 +/− 7 pg/mL) compared to control mIgG (118 +/− 15 pg/106 cells, N=3, p<0.004). Antibodies against CXCR2 and CXCR4 did not have any significant effect on TF induction. Measurement of MP TF activity mirrored the results described above. Using flow cytometry and an anti-CXCR3 antibody labeled with FITC, we found that 5% (± 0.5%) of monocytes expressed CXCR3 (N=3), which is consistent with the reported literature. Conclusions: These data suggest that induction of TF in monocytes by HIT antibody complexes is not mediated by the FcgRII receptor. This is contrary to the mechanism of platelet activation by these antibody complexes, which is an FcgRIIa dependent process. We found that TLR4 plays a role in HIT antibody complex mediated induction of TF in monocytes and blocking TLR4 led to a 30% decrease in TF activity. On the other hand, CXCR3 appeared to play a more significant role with blockade of CXCR3 leading to a 70% decrease in TF activity. Further characterization of the role of these receptors in HIT antibody complex mediated induction of TF expression in monocytes is required. We speculate that the extent of CXCR3 and TLR4 expression in monocytes may influence the susceptibility to developing thrombotic complications in HIT. Disclosures: No relevant conflicts of interest to declare.

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