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Enhanced tissue factor expression by blood eosinophils from patients with hypereosinophilia: a possible link with thrombosis.

Cugno M, Marzano AV, Lorini M, Carbonelli V, Tedeschi A - PLoS ONE (2014)

Bottom Line: Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis.TF gene expression was moderate in monocytes, Ct: 31.32 (29.82-33.49) and abundant in endothelial cells, Ct: 28.70 (27.79-29.57) and fibroblasts, Ct: 22.77 (19.22-25.05).The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milano, Italy; Medicina Interna, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milano, Italy.

ABSTRACT
Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis. In particular, eosinophils have been described as source of tissue factor (TF), the main initiator of blood coagulation; however, this aspect is still controversial. This study was aimed to evaluate whether TF expression varies in eosinophils isolated from normal subjects and patients with different hypereosinophilic conditions. Eosinophils were immunologically purified from peripheral blood samples of 9 patients with different hypereosinophilic conditions and 9 normal subjects. Western blot analysis and real-time polymerase chain reaction (RT-PCR) were performed to test eosinophil TF expression. For comparison, TF expression was evaluated in monocytes from blood donors and in human endothelial (ECV304) and fibroblast (IMR90) cell lines. Western blot analysis revealed a major band of 47,000 corresponding to native TF in homogenates of purified eosinophils with a higher intensity in the 9 patients than in the 9 controls (p<0.0001). According to RT-PCR cycle threshold (Ct), TF gene expression was higher in eosinophils from patients than in those from controls, median (range) 35.10 (19.45-36.50) vs 37.17 (35.33-37.87) (p = 0.002), and was particularly abundant in one patient with idiopathic hypereosinophilic syndrome and ischemic heart attacks (Ct: 19.45). TF gene expression was moderate in monocytes, Ct: 31.32 (29.82-33.49) and abundant in endothelial cells, Ct: 28.70 (27.79-29.57) and fibroblasts, Ct: 22.77 (19.22-25.05). Our results indicate that human blood eosinophils contain variable amounts of TF. The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.

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Related in: MedlinePlus

Representative cytocentrifuge smears of two high purity eosinophil preparations obtained from peripheral blood samples.May-Grünwald-Giemsa staining, original magnification: X 400 in the upper panels and X 1000 (immersion) in the lower panels.
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pone-0111862-g001: Representative cytocentrifuge smears of two high purity eosinophil preparations obtained from peripheral blood samples.May-Grünwald-Giemsa staining, original magnification: X 400 in the upper panels and X 1000 (immersion) in the lower panels.

Mentions: Leukocyte suspensions were obtained by dextran sedimentation of peripheral blood anticoagulated with 3.75% Na2EDTA (Sigma-Aldrich St Louis, Mo, USA) diluted 1∶2 in 0.9% sodium chloride. Dextran sedimentation (3 g D-Glucose, Sigma-Aldrich St Louis, Mo, USA; 3 g Dextran T500, Carl Roth Gmbh, Karlsruhe, Germany) lasted 90 minutes at room temperature. Twenty ml of leukocyte-enriched plasma were layered over 12 ml of a density gradient medium (sodium diatrizoate 9.1%; polysaccharide 5.7%; ρ = 1.077 g/ml, Fresenius Kabi, Oslo, Norway) in 50 ml conical tube and centrifuged at 600×g for 20 minutes at 20°C. The cell pellet containing eosinophils and neutrophils was collected and the contaminating red cells were eliminated by hypotonic ammonium chloride lysis solution (155 mM NH4Cl4, 10 mM KHCO3 and 0,1 mM Na2EDTA, Sigma-Aldrich St Louis, Mo, USA) for 10 minutes at 4°C. Contaminating neutrophils were removed using a magnetic-activated cell sorting system (Miltenyi Biotec Gmbh, Bergish Gladbach, Germany), containing a cocktail of biotin-conjugated monoclonal antibodies against CD2, CD14, CD16, CD19, CD56, CD123 and CD235a (Glycophorin A). Percentage purification of eosinophils recovered ranged from 95 to 99%, as assessed by differential count of 500 cells on May Grunwald Giemsa-stained cytocentrifuge smears (Figure 1). For protein extraction and western-blot analysis, 107 cells were used in each experiment. For RNA extraction, 3×106 cells were used in each experiment.


Enhanced tissue factor expression by blood eosinophils from patients with hypereosinophilia: a possible link with thrombosis.

Cugno M, Marzano AV, Lorini M, Carbonelli V, Tedeschi A - PLoS ONE (2014)

Representative cytocentrifuge smears of two high purity eosinophil preparations obtained from peripheral blood samples.May-Grünwald-Giemsa staining, original magnification: X 400 in the upper panels and X 1000 (immersion) in the lower panels.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222944&req=5

pone-0111862-g001: Representative cytocentrifuge smears of two high purity eosinophil preparations obtained from peripheral blood samples.May-Grünwald-Giemsa staining, original magnification: X 400 in the upper panels and X 1000 (immersion) in the lower panels.
Mentions: Leukocyte suspensions were obtained by dextran sedimentation of peripheral blood anticoagulated with 3.75% Na2EDTA (Sigma-Aldrich St Louis, Mo, USA) diluted 1∶2 in 0.9% sodium chloride. Dextran sedimentation (3 g D-Glucose, Sigma-Aldrich St Louis, Mo, USA; 3 g Dextran T500, Carl Roth Gmbh, Karlsruhe, Germany) lasted 90 minutes at room temperature. Twenty ml of leukocyte-enriched plasma were layered over 12 ml of a density gradient medium (sodium diatrizoate 9.1%; polysaccharide 5.7%; ρ = 1.077 g/ml, Fresenius Kabi, Oslo, Norway) in 50 ml conical tube and centrifuged at 600×g for 20 minutes at 20°C. The cell pellet containing eosinophils and neutrophils was collected and the contaminating red cells were eliminated by hypotonic ammonium chloride lysis solution (155 mM NH4Cl4, 10 mM KHCO3 and 0,1 mM Na2EDTA, Sigma-Aldrich St Louis, Mo, USA) for 10 minutes at 4°C. Contaminating neutrophils were removed using a magnetic-activated cell sorting system (Miltenyi Biotec Gmbh, Bergish Gladbach, Germany), containing a cocktail of biotin-conjugated monoclonal antibodies against CD2, CD14, CD16, CD19, CD56, CD123 and CD235a (Glycophorin A). Percentage purification of eosinophils recovered ranged from 95 to 99%, as assessed by differential count of 500 cells on May Grunwald Giemsa-stained cytocentrifuge smears (Figure 1). For protein extraction and western-blot analysis, 107 cells were used in each experiment. For RNA extraction, 3×106 cells were used in each experiment.

Bottom Line: Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis.TF gene expression was moderate in monocytes, Ct: 31.32 (29.82-33.49) and abundant in endothelial cells, Ct: 28.70 (27.79-29.57) and fibroblasts, Ct: 22.77 (19.22-25.05).The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, Università degli Studi di Milano, Milano, Italy; Medicina Interna, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milano, Italy.

ABSTRACT
Thrombotic risk is increased in eosinophil-mediated disorders, and several hypotheses have been proposed to link eosinophilia and thrombosis. In particular, eosinophils have been described as source of tissue factor (TF), the main initiator of blood coagulation; however, this aspect is still controversial. This study was aimed to evaluate whether TF expression varies in eosinophils isolated from normal subjects and patients with different hypereosinophilic conditions. Eosinophils were immunologically purified from peripheral blood samples of 9 patients with different hypereosinophilic conditions and 9 normal subjects. Western blot analysis and real-time polymerase chain reaction (RT-PCR) were performed to test eosinophil TF expression. For comparison, TF expression was evaluated in monocytes from blood donors and in human endothelial (ECV304) and fibroblast (IMR90) cell lines. Western blot analysis revealed a major band of 47,000 corresponding to native TF in homogenates of purified eosinophils with a higher intensity in the 9 patients than in the 9 controls (p<0.0001). According to RT-PCR cycle threshold (Ct), TF gene expression was higher in eosinophils from patients than in those from controls, median (range) 35.10 (19.45-36.50) vs 37.17 (35.33-37.87) (p = 0.002), and was particularly abundant in one patient with idiopathic hypereosinophilic syndrome and ischemic heart attacks (Ct: 19.45). TF gene expression was moderate in monocytes, Ct: 31.32 (29.82-33.49) and abundant in endothelial cells, Ct: 28.70 (27.79-29.57) and fibroblasts, Ct: 22.77 (19.22-25.05). Our results indicate that human blood eosinophils contain variable amounts of TF. The higher TF expression in patients with hypereosinophilic disorders may contribute to increase the thrombotic risk.

Show MeSH
Related in: MedlinePlus