scholarly journals Binding of gamma-glutamyl transferase to TLR-4 allows the activation of tissue factor expression in human monocytes

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
C Sanguinetti ◽  
V Scalise ◽  
T Neri ◽  
A Celi ◽  
R Pedrinelli
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Gamma Glutamyl Transferase ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Peripheral Blood Mononuclear ◽  
Gamma Glutamyl ◽  
Increased Risk ◽  
Glutamyl Transferase

Abstract Background Gamma-glutamyl transferase (GGT) plays a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the initiator of the clotting cascade. GGT may modulate TF expression, an assumption verified by previous studies carried out in human Peripheral Blood Mononuclear Cells (PBMCs). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic activity since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. Purpose To assess whether GGT-induced TF stimulation is a consequence of binding to Toll-Like Receptor (TLR)-4 and activation of NF-κB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from a leukemia patient, were incubated with hrGGT (0.5 ng/μl for PBMCs and 1ng/μl for THP-1). LPS-Rs (0.5 ng/μl for PBMCs and 1 ng/μl for THP-1), CLI-095 (3x10–6 M) and BAY-11-7082 (10–5 M) were used to block TLR-4 receptors, TLR4 signaling and NF-κB respectively. TF pro-coagulant activity (PCA) was assessed using of StartMax coagulometer and results were expressed in pg/ml after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110±70 to 510±43, n=7, p<0.01) and THP-1 cells (PCA from 170±64 to 460±80, n=15, p<0.001).In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72±17% n=4, p<0.01) a TLR-4 antagonist, CLI-095 (PCA:-74±34%, n=7, p<0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71±32%, n=7, p<0.001), a NF-κB inhibitor. Similar results were obtained in THP-1 cells [LPS-Rs: −76±15%, n=6, p<0.01; CLI-095: −100±6,6%, n=6, p<0.01; BAY-11-7082: −100±2,1%, n=6, p<0.01]. hrGGT activates NF-κB in hTLR4-positive HEK cell lines while doesn't induces effect in TLR4-negative HEK cells. Conclusions Besides confirming the cytokine-Like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells, these data identify for the first time the possible role of TLR-4 as the receptor of GGT and NfkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque. FUNDunding Acknowledgement Type of funding sources: None.

697 Binding of gamma-glutamyl transferase to toll-like receptor-4 allows tissue factor activation in monocytes

2021 ◽  
Vol 23 (Supplement_G) ◽  
Author(s):  
Chiara Sanguinetti ◽  
Valentina Scalise ◽  
Tommaso Neri ◽  
Alessandro Celi ◽  
Roberto Pedrinelli
Keyword(s):  
Western Blot ◽  
Tissue Factor ◽  
Cell Lines ◽  
Human Peripheral Blood ◽  
Gamma Glutamyl Transferase ◽  
Toll Like Receptor ◽  
Monocytic Cell ◽  
Gamma Glutamyl ◽  
Increased Risk ◽  
Glutamyl Transferase

Abstract Aims Gamma-glutamyl transferase (GGT) is well known to play a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of tissue factor (TF) gene expression, the principal initiator of the clotting cascade. GGT may potentially modulate TF expression, an assumption verified by previous studies carried out in human peripheral blood mononuclear cell (PBMC). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic properties since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation site. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. To assess whether GGT-induced TF stimulation is a consequence of binding to toll-like receptor (TLR)-4 and activation of NFkB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from an acute monocytic leukaemia patient, were incubated with hrGGT (0.5 ng/μL for PBMCs and 1 ng/µL for THP-1). LPS-Rs (0.5 ng/µL for PBMCs and 1 ng/µL for THP-1), CLI-095 (3 × 10−6M) and BAY-11-7082 (10−5 M) were used to block TLR-4 receptors, TLR4 signalling and NFkB respectively. TF protein concentration was determined by western blot analysis. TF pro-coagulant activity (PCA) was assessed through the use of Start Max coagulometer and results were expressed in pg/mL after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110 ± 70 to 510 ± 43, n = 7, P < 0.01) and THP-1 cells (PCA from 170 ± 64 to 460 ± 80, n = 15, P < 0.001), result confirmed by western blot. In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72 ± 17% n = 4, P < 0.01) a TLR-4 antagonist, CLI-095 (PCA: −74 ± 34%, n = 7, P < 0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71 ± 32%, n = 7, P < 0.001), a NF-kB inhibitor. Similar results were obtained in THP-1 cells (LPS-Rs: −76 ± 15%, n = 6, P < 0.01; CLI-095: −100 ± 6,6 %, n = 6, P < 0.01; BAY-11-7082: −100 ± 2,1%, n = 6, P < 0.01). hrGGT activates NFkB in hTLR4-positive HEK cell lines while does not induces effect in TLR4-negative HEK cell lines. Conclusions These data confirm the cytokine-like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells. Furthermore, it is highlighted for the first time the possible role of TLR-4 as the receptor of GGT and NFkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the consistent association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the silent atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque.

scholarly journals Mechanisms of Inflammasome Activation by Vibrio cholerae Secreted Toxins Vary with Strain Biotype

2015 ◽  
Vol 83 (6) ◽  
pp. 2496-2506 ◽  
Author(s):  
Jessica Queen ◽  
Shivani Agarwal ◽  
Jazel S. Dolores ◽  
Christian Stehlik ◽  
Karla J. F. Satchell
Keyword(s):  
Vibrio Cholerae ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Inflammasome Activation ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Peripheral Blood Mononuclear ◽  
Content Type ◽  
Murine Macrophages ◽  
El Tor

Activation of inflammasomes is an important aspect of innate immune responses to bacterial infection. Recent studies have linkedVibrio choleraesecreted toxins to inflammasome activation by using murine macrophages. To increase relevance to human infection, studies of inflammasome-dependent cytokine secretion were conducted with the human THP-1 monocytic cell line and corroborated in primary human peripheral blood mononuclear cells (PBMCs). Both El Tor and classical strains ofV. choleraeactivated ASC (apoptosis-associated speck-like protein-containing a CARD domain)-dependent release of interleukin-1β (IL-1β) when cultured with human THP-1 cells, but the pattern of induction was distinct, depending on the repertoire of toxins the strains produced. El Tor biotype strains induced release of IL-1β dependent on NOD-like receptor family pyrin domain-containing 3 (NLRP3) and ASC due to the secreted pore-forming toxin hemolysin. Unlike in studies with mouse macrophages, the MARTX toxin did not contribute to IL-1β release from human monocytic cells. Classical biotype strains, which do not produce either hemolysin or the MARTX toxin, activated low-level IL-1β release that was induced by cholera toxin (CT) and dependent on ASC but independent of NLRP3 and pyroptosis. El Tor strains likewise showed increased IL-1β production dependent on CT when the hemolysin gene was deleted. In contrast to studies with murine macrophages, this phenotype was dependent on a catalytically active CT A subunit capable of inducing production of cyclic AMP and not on the B subunit. These studies demonstrate that the induction of the inflammasome in human THP-1 monocytes and in PBMCs byV. choleraevaries with the biotype and is mediated by both NLRP3-dependent and -independent pathways.

scholarly journals Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2516-2525 ◽  
Author(s):  
K Meszaros ◽  
S Aberle ◽  
R Dedrick ◽  
R Machovich ◽  
A Horwitz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Mononuclear Phagocytes ◽  
Factor Vii ◽  
Peripheral Blood Mononuclear ◽  
Recombinant Fragment

Abstract Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

Recombinant Tissue Factor Pathway Inhibitor Enhances the Binding of Factor Xa to Human Monocytes

2001 ◽  
Vol 85 (05) ◽  
pp. 830-836
Author(s):  
Anguo Li ◽  
Alvin Chang ◽  
Glenn Peer ◽  
Tze-Chen Wun ◽  
Fletcher Taylor
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Factor Xa ◽  
Human Monocytes ◽  
Factor X ◽  
Peripheral Blood Mononuclear ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a kunitz-type inhibitor of activated factor X (Xa). TFPI was reported to mediate Xa binding to a few of carcinoma cell lines. In this study it was observed that the Xa activity associated with human peripheral blood mononuclear cells (PBMC) incubated with Xa in the presence of recombinant TFPI (rTFPI) was much higher than with Xa alone. Xa activity on PBMC was also observed after whole blood was incubated with pre-formed Xa/TFPI complex. Further studies with flow cytometric analysis demonstrate that rTFPI enhances the binding of Xa to human monocytes. Western blot analysis showed that rTFPI was cleaved into a few of fragments after its incubation with monocytes either in the presence or absence of Xa. Based on these results and the observations reported by others, we speculate that Xa/TFPI complex may bind to human monocytes by a yet unidentified mechanism. The recovery of Xa activity from Xa/TFPI complex on PBMC may be related to the cleavage of rTFPI by Xa and/or monocyte proteases. This observation suggests a new mechanism by which monocytes become procoagulant in some pathological conditions in addition of the well known tissue factor expression on proinflammatic monocytes.

TNF-related apoptosis-inducing ligand (TRAIL) blocks osteoclastic differentiation induced by RANKL plus M-CSF

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 2044-2050 ◽  
Author(s):  
Giorgio Zauli ◽  
Erika Rimondi ◽  
Vanessa Nicolin ◽  
Elisabetta Melloni ◽  
Claudio Celeghini ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Mononuclear Cells ◽  
Mapk Pathway ◽  
Human Peripheral Blood ◽  
Tartrate Resistant Acid Phosphatase ◽  
Cell Models ◽  
Monocytic Cell ◽  
Monocytic Cell Line ◽  
Tnf Superfamily ◽  
Osteoclastic Differentiation

Abstract The role of the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor kappa B ligand (RANKL) in promoting the differentiation of osteoclasts has been extensively characterized. In this study, we have investigated the effect of TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of cytokines, in osteoclastogenesis, by using human peripheral blood mononuclear cells and the RAW264.7 murine monocytic cell line. Both cell models differentiate into osteoclast-like cells in presence of RANKL plus macrophage-colony-stimulating factor (M-CSF), as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Unexpectedly, when added in culture in combination with RANKL plus M-CSF, TRAIL inhibited osteoclastic differentiation in both cell models. To investigate the molecular mechanism underlining such inhibitory activity, we analyzed the effect of TRAIL on the mitogen-activated protein kinases (MAPKs) pathways, which play a key role in osteoclastogenesis. Treatment with RANKL plus M-CSF activated both the ERK1/2 and p38/MAPK pathways, which are essential for proliferation and differentiation of preosteoclasts, respectively. Of note, the addition of TRAIL to RANKL plus M-CSF did not affect ERK1/2 but it profoundly inhibited p38/MAPK phosphorylation. Thus, our data demonstrate that TRAIL blocks osteoclastic differentiation and suggest that inhibition of the p38/MAPK pathway by TRAIL likely plays an important role in this process. (Blood. 2004;104:2044-2050)

scholarly journals Peptidoglycan from Staphylococcus aureus Induces Tissue Factor Expression and Procoagulant Activity in Human Monocytes

2002 ◽  
Vol 70 (6) ◽  
pp. 3033-3039 ◽  
Author(s):  
Eva Mattsson ◽  
Heiko Herwald ◽  
Lars Björck ◽  
Arne Egesten
Keyword(s):  
Staphylococcus Aureus ◽  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Procoagulant Activity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Extrinsic Pathway ◽  
Peripheral Blood Mononuclear ◽  
Human Sepsis ◽  
Reverse Transcription Pcr

ABSTRACT Staphylococcus aureus is one of the most significant pathogens in human sepsis and endocarditis. S. aureus can initiate blood coagulation, leading to the formation of microthrombi and multiorgan dysfunction in sepsis, whereas in endocarditis the bacterium induces fibrin clots on the inner surface of the heart, so-called endocardial vegetations. In the present study, we show that live and heat-killed S. aureus bacteria are potent inducers of procoagulant activity in human peripheral blood mononuclear cells. Furthermore, purified peptidoglycan, the main cell wall component of S. aureus, induced procoagulant activity in mononuclear cells in a concentration-dependent fashion. The procoagulant activity in these cells was dependent on expression of tissue factor, since antibodies to tissue factor inhibited the effect of peptidoglycan. In mononuclear cells stimulated with peptidoglycan, reverse transcription-PCR showed tissue factor gene expression, and the gene product was detected by enzyme-linked immunosorbent assay. Finally, flow cytometry identified tissue factor at the surface of CD14-positive monocytes. Peptidoglycan is known to induce proinflammatory cytokine production in monocytes. The present investigation shows that peptidoglycan also activates the extrinsic pathway of coagulation by inducing the expression of tissue factor in these cells. This mechanism helps to explain the procoagulant activity, which plays such an important role in the pathogenicity of severe S. aureus infections.

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