Protein Disulfide Isomerase Plays An Important Role in Beta2 Integrin-Mediated Neutrophil Recruitment During Vascular Inflammation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 17-17
Author(s):  
Eunsil Hahm ◽  
Sungjin Huh ◽  
Snezna Rogelj ◽  
Jaehyung Cho
Keyword(s):  
Protein Disulfide Isomerase ◽  
Conflicts Of Interest ◽  
Vascular Inflammation ◽  
Neutrophil Recruitment ◽  
Intercellular Adhesion Molecule ◽  
Tnf Alpha ◽  
Neutrophil Adhesion ◽  
Disulfide Isomerase ◽  
Static Conditions ◽  
Protein Disulfide

Abstract Abstract 17 Cell surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, plays an important role in regulating integrin-mediated cellular functions. Although surface PDI has been reported to regulate L-selectin shedding on neutrophils, its role in neutrophil recruitment during vascular inflammation has not been explored. In this paper, we present novel findings on how neutrophil PDI regulates neutrophil recruitment into the site of vascular inflammation. Using real-time microscopy with cell-impermeable inhibitors for PDI (bacitracin or function-blocking antibodies), we demonstrated that inhibition of PDI reduces adhesion of human blood neutrophils to TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under venous shear. There was no additive effect on neutrophil adhesion in a combination of blocking anti-PDI and anti-beta2 antibodies, suggesting that surface PDI regulates beta2 integrin-mediated neutrophil adhesion to TNF-alpha-activated HUVECs under shear. Further, bacitracin and blocking anti-PDI antibodies diminished alphaMbeta2 integrin-mediated neutrophil adhesion to intercellular adhesion molecule-1 (ICAM-1)-coated surfaces under static conditions. When PDI gene is knocked down in neutrophil-like HL60 cells by shRNA, alphaMbeta2 activation and cell adhesion to TNF-alpha-activated HUVECs under shear were significantly reduced. High resolution confocal microscopy and immunoprecipitation assay revealed that surface PDI interacts with alphaMbeta2 integrin and that such interaction was enhanced by neutrophil activation and inhibited by bacitracin, a thiol isomerase inhibitor. Exogenous PDI equivalently bound to CHO cells stably expressing alphaMbeta2 or alphaLbeta2 when those cells were treated with Mn2+, whereas PDI binding was significantly reduced when the active site CGHC residues on PDI were mutated. Using multi-channel fluorescence intravital microscopy, we demonstrated that inhibition of PDI by infusion of PDI inhibitors impairs stable adhesion of neutrophils to the TNF-alpha-inflamed cremaster muscle venule wall in living mice. These findings suggest the potentially fundamental but poorly understood role for surface PDI in the regulation of beta2 integrin function in neutrophils. Therefore, our results provide the first evidence that inhibition of PDI may be a therapeutic target for the neutrophil-mediated vascular inflammation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Extracellular protein disulfide isomerase regulates ligand-binding activity of αMβ2 integrin and neutrophil recruitment during vascular inflammation

Blood ◽  
2013 ◽  
Vol 121 (19) ◽  
pp. 3789-3800 ◽  
Author(s):  
Eunsil Hahm ◽  
Jing Li ◽  
Kyungho Kim ◽  
Sungjin Huh ◽  
Snezna Rogelj ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Vascular Inflammation ◽  
Binding Activity ◽  
Neutrophil Recruitment ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
Key Points ◽  
Protein Disulfide ◽  
Ligand Binding Activity ◽  
Neutrophil Surface

Key Points This work is the first identification of a neutrophil surface thiol isomerase regulating adhesive function of αMβ2 integrin. PDI is required for neutrophil recruitment during vascular inflammation and its isomerase activity is critical for the regulatory effect.

Platelet ERp57 Is Required for Hemostasis and Thrombosis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2168-2168
Author(s):  
Lu Wang ◽  
Yi Wu ◽  
Junsong Zhou ◽  
Syed S. Ahmad ◽  
Bulent Mutus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Protein Disulfide Isomerase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Abstract 2168 Several members of the protein disulfide isomerase family of enzymes are important in platelet function and in thrombosis. Platelet protein disulfide isomerase (PDI) has been shown to have an important role in platelet function but is reported to not be required for thrombus formation in vivo. A novel platelet PDI called ERp57 mediates platelet aggregation but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis we generated a megakaryocyte/platelet specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times, and defective activation of the αIIbβ3 integrin and platelet aggregation. The aggregation defect was corrected by addition of exogenous ERp57 implicating surface ERp57 in platelet aggregation. Platelet surface ERp57 protein and activity increased substantially with platelet activation. We conclude that platelet-derived ERp57 is required for hemostasis and thrombosis and platelet function. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activation of the Mineralocorticoid Receptor Stimulates Protein Disulfide Isomerase: Role in Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1351-1351
Author(s):  
Alexandra Lozano ◽  
Christopher Vega ◽  
Yaritza Inostroza-Nieves ◽  
Lorena Rivera González ◽  
Pablo J. López ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Protein Disulfide Isomerase ◽  
Mineralocorticoid Receptor ◽  
Conflicts Of Interest ◽  
Hematological Parameters ◽  
Control Diet ◽  
Mrna Levels ◽  
Disulfide Isomerase ◽  
Qrt Pcr ◽  
Protein Disulfide

Abstract Activation of the mineralocorticoid receptor (MR), a critical component of the Renin-Angiotensin-Aldosterone (ALDO)-System (RAAS), has been shown to play an important role in inflammatory and vascular endothelial responses in addition to its well-described effects on sodium and water homeostasis. Activationof endothelial cells leads to, among other factors, increased endothelin-1 (ET-1) and protein disulfide isomerase (PDI) release. PDI and ET-1 contribute to vascular inflammation and are increased in patients with SCD and sickle transgenic mouse models. The MR is a member of the steroid family of nuclear receptors and transcription factors that upon activation binds to hormone response elements of edn1, the gene for ET-1, leading to increased ET-1 expression.In vivo, blockade of MR has been shown to reduce circulating ET-1 levels and ET-1 mRNA expression. However, the role of MR in SCD is unclear. We hypothesized that MR blockade in sickle transgenic mice would reduce PDI activity and improve hematological parameters and inflammation. We first studied EA.hy926 (EA) cells, a human endothelial cell line that expresses MR. We incubated EA cells with ALDO (10-8 M), an MR agonist, for 24 hr and observed a rise in PDI mRNA levels by qRT-PCR (P<0.01, n=5), an event that was blocked by pre-incubation of EA cells with 1 μM canrenoic acid (CA), an MR antagonist (P<0.05, n=5). We then measured PDI activity in the supernatant of ALDO-stimulated EA cells using a Di-E-GSSH fluorescent marker and observed a rise in PDI activity following ALDO (10-8 M) when compared to vehicle treatment (P<0.05; n=5). To test the in vivo effects of MR activation, we studied Berkeley Sickle Transgenic (BERK) mice that were randomized to receive either normal rodent chow or chow containing eplerenone (156 mg/kg per day), an MR antagonist (MRA), for 14 days. We observed significantly lower plasma PDI activity in mice treated with MRA than those on regular chow (63.7 ± 8.7 control diet to 47.9 ± 2.4 eplerenone, Relative Fluorescence Units (RFU); P<0.005, n=6 and 9, respectively). Treatment with MRA was associated with reduced plasma ET-1 and myeloperoxidase (MPO) levels in BERK mice. We also studied RBC Gardos channel activity in these mice and observed a significant reduction in clotrimazole-sensitive K+ efflux following MR blockade (2.49±0.5 control and 1.37±0.3 mmol/1013 cells x hr; P<0.04 n= 5 and 7 respectively). Consistent with these results, MR blockade was associated with increases in both erythrocyte MCV (41.3±2.5 vs 47.4±1.1 fL, P<0.03, n=7) and reticulocyte MCV (53.6.3±2.8 vs 60.1±0.6 fL, P<0.02, n=7). We also studied gene expression by qRT-PCR in heart tissue from these mice and observed that MR blockade reduced mRNA expression of: ET-1 (0.654 ± 0.233, P<0.05, n=5 and n=7); PDI (0.546 ± 0.063, P<0.01, n=5 and n=7); and Tumor Necrosis Factor Receptor Superfamily Member 1A mRNA (0.464 ± 0.061, P<0.01, n=5 and n=7). Thus, our results suggest a novel role for RAAS and, in particular, MR activation in SCD. Disclosures No relevant conflicts of interest to declare.

Endothelial-Specific Knockdown Of Caveolin-1 Lowers Circulating Protein Disulfide Isomerase Levels and Is Associated With Changes In Erythrocyte Volume Homeostasis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 12-12
Author(s):  
Josue A. Benabe-Carlo ◽  
Jose R Romero ◽  
Enrique D. Machado-Fiallo ◽  
Arelys Ramos-Rivera ◽  
Gregory N. Prado ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Disulfide Isomerase ◽  
Conflicts Of Interest ◽  
Mean Cell Volume ◽  
Distribution Width ◽  
Disulfide Isomerase ◽  
Erythrocyte Volume ◽  
Caveolin 1 ◽  
Protein Disulfide

Abstract We have recently reported that Endothelin-1 (ET1), a potent vasoconstrictor peptide, is implicated in the pathophysiology of Sickle Cell Disease (SCD) via increased circulating Protein Disulfide Isomerase (PDI) activity (Prado, 2013 FASEB J). PDI is a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, catalyzes disulfide interchange reactions in the plasma membrane, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions as commonly observed in SCD. In erythrocytes, ET1 stimulates PDI activity via activation of ET1 receptor B (ETRB). However, the precise mechanisms by which ET1 leads to increases in PDI are not entirely clear. There is evidence that activation of endothelial cells leads to increased PDI secretion and that ETRBs form a complex with caveolin-1 (CAV1) within caveolae to mediate ET1’s cellular effects. We tested the hypothesis that reduction of CAV1 would alter PDI secretion. We studied the in vivo effects of endothelial-specific CAV1 knockdown on circulating PDI activity in mice. We optimized conditions to measure circulating PDI using fluorescently labeled GSSG conversion to GSH. We now report that circulating plasma PDI levels were significantly decreased in CAV1 knockdown mice when compared to wild-type littermates (WT) (7.44±0.70 vs 10.93±2.66, n=7, P<0.05). In addition and consistent with our report showing a role for PDI in erythrocyte volume regulation, we also observed lower cell hemoglobin concentration mean (CHCM) and hemoglobin distribution width (HDW) that was associated with increased erythrocyte and reticulocyte mean cell volume (MCV) in blood from CAV1 knockdown mice when compared to WT (n=13 and n=19, respectively, P<0.005). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from these mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from CAV1 knockdown mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). We then studied the effects of ET1 on PDI secretion from human endothelial cells. We detected PDI and ETRB by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We observed that incubation of EA cells for 60 mins with 10-7 M ET1 was associated with increased extracellular PDI activity (15.97±7.22 to 34.07±8.89 [RFU/mg protein], n=3, P<0.011) that was sensitive to preincubation with BQ788, a specific ETRB receptor antagonist (15.97±7.22 to 7.97±3.25 (RFU/mg protein), n=3, P<0.02). Similar increases in PDI were observed when cells were treated with the specific ETRB agonist, IRL1620 (1143±137 to 1593 207 RFU/mg protein). In addition, PDI siRNA knockdown was associated with reduced ET1-stimulated PDI activity when compared to scrambled siRNA transfected cells (1731±147 to 757±141 RFU, n=2). We then tested the effects of methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted IRL1620–stimulated PDI response (288±40 to 171±14 RFU/mg protein, n=3, P<0.025). We also characterized the effects of ET1 on PDI expression in EA cells, using quantitative RT-PCR with ABI TaqMan probes and β-actin as an endogenous control and observed that stimulation of EA cells with 10-8 M ET1 for 4 hr was associated with increased PDI mRNA expression levels that were 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Thus our results provide evidence for a heretofore unrecognized role of endothelial specific CAV1 in erythrocyte volume and circulating PDI levels. Supported by NIH R01HL090632 (AR) and R01HL104032 (LHP). Disclosures: No relevant conflicts of interest to declare.

β2-integrin activity: the role of thiols

Blood ◽  
2013 ◽  
Vol 121 (19) ◽  
pp. 3779-3780 ◽  
Author(s):  
Alexander Zarbock
Keyword(s):  
Disulfide Bonds ◽  
Protein Disulfide Isomerase ◽  
Neutrophil Recruitment ◽  
Regulatory Effect ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
Β2 Integrin ◽  
Protein Disulfide

In this issue of Blood, Hahm and colleagues identify the extracellular protein disulfide isomerase (PDI) as an essential regulator of the adhesiveness of the β2-integrin macrophage-1 antigen (Mac-1) on neutrophils.1 In the absence of PDI, Mac-1–dependent neutrophil adhesion and crawling is reduced in vivo. Rescue experiments with exogenous PDI showed that the isomerase activity of extracellular PDI is critical for its regulatory effect on neutrophil recruitment. This intriguing finding suggests that disulfide bonds in Mac-1 regulate integrin activity and neutrophil recruitment.

Neutrophil Akt2 Plays a Critical Role In Heterotypic Neutrophil-Platelet Interactions During Vascular Inflammation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 321-321
Author(s):  
Jing Li ◽  
Kyungho Kim ◽  
Eunsil Hahm ◽  
Victor R. Gordeuk ◽  
Nissim Hay ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Vascular Inflammation ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Microscopic Analysis ◽  
Neutrophil Recruitment ◽  
Neutrophil Adhesion ◽  
Inflammatory Conditions ◽  
Healthy Donors

Abstract Platelet-leukocyte interactions on activated endothelial cells play important roles in mediating pathological thrombosis and inflammation. Heterotypic platelet-leukocyte aggregation is mediated by the interaction of two crucial receptors and counter receptors; P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. In spite of extensive understanding of receptor-counter receptor interactions, it remains unclear how heterotypic cell-cell interactions are regulated under thrombo-inflammatory conditions. Using real-time fluorescence intravital microscopic analysis of Akt isoform-specific knockout (KO) mice, we have demonstrated that Akt2, but not Akt1 or Akt3, plays an important role in neutrophil adhesion to the site of TNF-alpha-induced vascular inflammation. Further, heterotypic platelet-neutrophil interactions on the activated endothelium were markedly reduced in Akt2 but not Akt1 or Akt3 KO mice. Studies with chimeric mice generated from bone marrow transplants on wild-type and Akt2 KO mice revealed that hematopoietic but not endothelial cell Akt2 regulates neutrophil recruitment and platelet-neutrophil interactions during vascular inflammation. Using in vitro reconstituted systems in which platelets and neutrophils were treated with an Akt2 specific inhibitor or cells were isolated from WT and Akt KO mice, we observed that both platelet and neutrophil Akt2 play an important role in platelet-neutrophil aggregation under shear conditions. In particular, neutrophil Akt2 was critical for membrane translocation, activation, and adhesive function of alphaMbeta2 integrin. We found that the basal phosphorylation levels of Akt isoforms are significantly increased in neutrophils and platelets of patients with sickle cell disease (SCD), which is an inherited hematological disorder with vascular inflammation and occlusion. Also, SCD patients’ neutrophils show increased alphaMbeta2 integrin activation in the absence of an agonist, in comparison with healthy donors’ cells. Inhibition of Akt2 dose-dependently reduced heterotypic aggregation of patients’ neutrophils and platelets in vitro and inhibited neutrophil adhesion and neutrophil-platelet aggregation in SCD mice, thereby improving blood flow. Our results provide important genetic and pharmacologic evidence that neutrophil Akt2 regulates alphaMbeta2 integrin function and thus plays a critical role during neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document