scholarly journals Heme Induces Significant Neutrophil Adhesion in Vitro Via an Nfκb-Dependent Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3610-3610
Author(s):  
Lediana I. Miguel ◽  
Lidiane S. Torres ◽  
Wilson A. Ferreira ◽  
Fernanda C. Z. Fabris ◽  
Flavia C. Leonardo ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Cytochalasin D ◽  
Human Neutrophils ◽  
Neutrophil Adhesion ◽  
Integrin Subunit ◽  
Adhesive Properties ◽  
Tnf Α ◽  
Dependent Pathway

Abstract Background: Intravascular hemolysis, a major complication of sickle cell anemia and malaria among other diseases, incurs the release of excessive quantities of hemoglobin and heme from red blood cells. If not adequately sequestered by hemoglobin- and heme-binding proteins, these molecules may incur oxidative stress, thrombosis and cellular activation. Heme is now recognized as a red cell DAMP (damage-associated molecular pattern) that stimulates inflammasome formation in macrophages and can induce neutrophil extracellular trap release under certain circumstances. Aim: This study evaluated the in vitro effects of heme on the adhesive properties of human neutrophils. Methods: Neutrophils were separated from the peripheral blood of healthy individuals and their adhesion in the presence/absence of heme was compared by static adhesion assays using myeloperoxidase, for quantification of cell adhesion (30 min, 37oC, 5% CO2). Results: Heme (50 µM) significantly increased the adhesion of neutrophils to fibronectin (FN) and to recombinant ICAM-1 (an endothelial ligand), when compared to non-treated neutrophils (43.7±4.6%; 10.9±21.4 %, respectively, n=5, P<0.001 for FN and 38.1±3.6%; 6.9±0.8%, respectively, n=6, P<0.001 for ICAM-1). Interestingly, heme induced neutrophil adhesion even more efficiently than the potent pro-inflammatory cytokine, TNF-α (200 ng/ml) (35.5±5.2% adhesion to FN, n=6, P<0.05). Furthermore, inhibition of the NFκB transcription factor with the pharmacological inhibitor, BAY 11-7082 (20 μM), abolished heme-stimulated neutrophils adhesion to FN (reduced from 41.8±6.7% to 7.4±0.5%. n=6; P<0.001). Flow cytometry demonstrated that while TNF-α significantly increases the expression of the Mac-1 integrin subunit, CD11b (data not shown, P<0.05), but not the LFA-1 integrin subunit CD11a (data not shown, P>0.05), on the surface of neutrophils, heme did not augment CD11b or CD11a expression (P>0.05). In contrast, heme significantly augmented the active conformations of these two β2 integrin subunits, as demonstrated by epitope-specific antibodies (CD11b; heme: 520.5±51.8; basal 123.7±16.7 MFI, n=10, P<0.001 and CD11a; heme: 69.8±3.9; basal: 43.8±2.0 MFI, n=6, P>0.001). Inhibition of NFκB translocation with BAY 11-7082 (20 μM) significantly decreased the activity of the LFA-1 integrin on the surface of neutrophils after heme stimulation (reduced to 47.6±4.2 MIF, n=6; P<0.05). To assess whether heme-induced neutrophil adhesive properties are mediated by cytoskeletal rearrangements, we evaluated the effects of cytochalasin D (0.5 μg/ml), an inhibitor of actin polymerization. While cytochalasin D inhibited TNF-α-induced neutrophils adhesion (data not shown, P<0.05), this compound did not significantly alter the adhesive properties of heme-stimulated neutrophils to FN (heme: 22.8±2.3%, p<0.001; cytochalasin D: 22.5±2.0%, p=0.4; n=6). Pre-incubation of heme-stimulated neutrophils with the antioxidants, ascorbic acid (120 µM; 3.5 hours) and α-tocopherol (1mM; 15 min), reduced their adhesion to FN by 14.4±9.2% and 46.5±5.5% respect. n=6, p<0.05). Conclusion: We therefore demonstrate, herein, that heme is a potent activator of neutrophil adhesive properties, increasing the ligand affinity of the β2 integrins via a mechanism that is apparently mediated by an NFkB-dependent pathway. The mechanism of neutrophil activation appears to differ from that stimulated by TNF-α and may involve, in part, the generation of reactive oxygen species. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in the vaso-occlusive process in sickle cell disease and other vascular inflammatory processes, our findings further support the idea that cell-free heme represents a major therapeutic target in the hemolytic diseases. Disclosures No relevant conflicts of interest to declare.

The Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers, In Vitro, Is Mediated by the Mac-1, LFA-1 and VLA-4 Integrins.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2264-2264 ◽  
Author(s):  
Andreia A. Canalli ◽  
Renata F. Proenca ◽  
Sara T.O. Saad ◽  
Nicola Conran ◽  
Fernando F. Costa
Keyword(s):  
Endothelial Cells ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Umbilical Vein ◽  
In Vivo Studies ◽  
Integrin Subunit ◽  
Cell Disease ◽  
Inflammatory Conditions ◽  
Tnf Α

Abstract Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. In vivo studies suggest that adherent leukocytes capture sickle erythrocytes in the microcirculation and in vitro studies demonstrate an increased ability of SCD neutrophils (neu) to adhere to fibronectin, endothelial cells and endothelial proteins. Previous studies suggest that the expressions of the major neu integrins, CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) may only be upregulated on the surface of SCD neu following their stimulation, indicating that alterations in integrin function (affinity or avidity) contribute to alter SCD neu adhesion. The objective of this study was to identify the integrins responsible for altered SCD neu adhesion. Neus were isolated from the peripheral blood of healthy controls and SCD individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106cells/ml in RPMI) to cultured human umbilical vein endothelial cells (HUVEC) at confluence was assessed using static adhesion assays (30min, 37°C, 5%CO2). Neus from SCD patients demonstrated a significantly greater adhesion to HUVEC than control neus (20.2±2.8% compared to 11.2±1.0%; n≥7; p&lt;0.03; Mann Whitney test). Subsequently, cells were co-incubated with adhesion molecule-blocking monoclonal antibodies (mAbs) during assays. Control neu adhesion to HUVEC was significantly inhibited by the anti-CD11b mAb (6.7±1.5%;n=6; P&lt;0.05, paired t test), but not by mAbs against CD11a, the VLA-4-integrin subunit, CD49d, or a non-specific negative control mAb (neg control) (data not shown). In contrast, the adhesion of SCD neus to HUVEC was significantly inhibited by both the anti-CD11a and the anti-CD11b mAbs (20.2±2.8% reduced to 11.4±1.2% and 9.1±1.5%; n=9; P&lt;0.01 and P&lt;0.001, respect.). Interestingly, a mAb against CD49d was also found to significantly decrease SCD neu adhesion to HUVEC (10.4±1.1%; n=9; P&lt;0.01), while the neg control mAb did not significantly affect SCD neu adhesion (data not shown). Following the stimulation of HUVEC with TNF-α (10 ng/ml) (3h, 37°C, 5%CO2) to simulate an endothelial layer under inflammatory conditions, the adhesions of control and SCD neus were increased but statistically similar (38.4±2.9% and 34.4±5.0%; n≥4, respect.). Under these conditions anti-CD11a and CD11b mAbs significantly inhibited control neu adhesion to HUVEC (reduced to 28.8±2.9% and 19.6±4.6%; n=4; P&lt;0.01 and P&lt;0.05, respect.). In contrast, SCD neu adhesion to HUVEC was significantly inhibited by mAbs for CD11a (19.5±2.6%; n=6; p&lt;0.01) and CD11b (15.2±2.0%; n=6; p&lt;0.001). The anti-CD49d, but not the neg control mAb, also significantly decreased SCD neu adhesion to TNF-α-stimulated HUVEC (19.5±3.7%; n=6; p&lt;0.05). In conclusion, data indicate that control neu adhesion to endothelial cells appears mainly to be mediated by the Mac-1 (CD11b/18) integrin with a contribution from the LFA-1 integrin (CD11a/18) under inflammatory conditions. In contrast, SCD neu adhesion to endothelium (under both basal and stimulated conditions), at least in vitro, appears to be mediated by the Mac-1 and LFA-1 integrins and, interestingly, by VLA-4 (CD49d/CD29), an integrin found expressed at low levels on neus during certain inflammatory conditions. We speculate that alterations in the affinity/ avidity of these molecules contribute to SCD neu adhesion. Approaches to inhibit the adhesion of all three integrins may be important for preventing leukocyte adhesion to the vascular endothelium and, in turn, vaso-occlusion.

Retention of leukocytes in capillaries: role of cell size and deformability

1990 ◽  
Vol 69 (5) ◽  
pp. 1767-1778 ◽  
Author(s):  
G. P. Downey ◽  
D. E. Doherty ◽  
B. Schwab ◽  
E. L. Elson ◽  
P. M. Henson ◽  
...  
Keyword(s):  
Cell Size ◽  
Dynamic Equilibrium ◽  
Human Neutrophils ◽  
Biophysical Properties ◽  
Adhesive Properties ◽  
Pulmonary Capillaries ◽  
Size Discrepancy ◽  
Capillary Bed ◽  
Filtration Properties

Leukocytes within the circulation are in dynamic equilibrium with a marginated pool, thought to reside mainly within the pulmonary capillaries. The size discrepancy between the mean diameter of circulating leukocytes (6-8 microns) and that of the pulmonary capillaries (approximately 5.5 microns) forces the cells to deform in order to transit the capillary bed. Consequently, we investigated the hypothesis that the biophysical properties of cell size and deformability determined differential leukocyte retention in the lung. Comparison of the filtration properties of human neutrophils, lymphocytes, monocytes, platelets, and erythrocytes through polycarbonate filters (5-micron pore diameter) revealed that the largest leukocytes (neutrophils and monocytes) were retained to the greatest extent and the smaller cells (lymphocytes and platelets) the least. Undifferentiated HL-60 cells, of greater diameter than their differentiated counterparts, were also retained to a greater extent, confirming that cell size was one important determinant of retention in these model capillaries. However, compared with neutrophils, which are of similar diameter, monocytes were retained to a greater extent, suggesting that monocytes might be less deformable than neutrophils. To test this hypothesis, deformability was measured directly using the cell poker. Monocytes were found to be the stiffest, neutrophils the softest, and lymphocytes intermediate. Glutaraldehyde treatment of neutrophils markedly increased their stiffness and decreased their ability to transit the pores of the filters in vitro and the pulmonary microvasculature of rabbits without changing their adhesive properties or size. These observations support the hypothesis that biophysical properties of leukocytes (size and deformability) determine in part their ability to transit the pulmonary capillaries and may determine the magnitude of their marginated pools.

P0914NEUTROPHIL EXTRACELLULAR TRAPS FORMATION IN HEMODIALYSIS PATIENTS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Adi Litmanovich ◽  
Khaled Khazim ◽  
Kamal Hassan ◽  
Batya Kristal
Keyword(s):  
Calcium Ionophore ◽  
Morbidity And Mortality ◽  
Human Neutrophils ◽  
Dialysis Session ◽  
Ionophore A23187 ◽  
Arterial Line ◽  
Sod Activity ◽  
Dependent Pathway ◽  
Normal Controls

Abstract Background and Aims Hemodialysis (HD) patients suffer from devastatingly high rates of morbidity and mortality due to infections. Neutrophils isolated from HD patients were shown in the past to exhibit impaired phagocytosis in a mechanism yet to be completely elucidated. In 2004, Brinkmann et al. were the first to describe a new form of cell death which they termed Neutrophil Extracellular Trap Formation, or NETosis, in which neutrophils expulse batches of DNA and proteins in response to bacterial or chemical stimuli in order to trap and remove the stimulus. NETosis is further divided into two pathways, NADPH oxidase (NOX)-dependent and NOX-independent, induced in vitro by phorbol 12-myristate 13-acetate (PMA) and Calcium Ionophore (CI), respectively. In this research, we aim to assess the capacities of HD neutrophils to engage in NETosis, hypothesizing they might be diminished similarly to their phagocytic capacities, and to elucidate the underlying mechanism behind this impairment. Method Neutrophils were isolated from whole venous blood of normal controls and from the arterial line of HD patients before the onset of a dialysis session using EasySepTM direct human neutrophils isolation kit. Then, NETosis was induced with either PMA, Calcium Ionophore A23187 or Phosphate-buffered saline (PBS) as negative control. cfDNA released from the cells was quantified by measuring SYTOXTM-green nucleic acid stain fluorescence levels in the supernatant after stimulation using Elisa plate reader and morphological analysis was done under fluorescence microscope. Reactive oxygen species levels were quantified using flow cytometry and superoxide dismutase (SOD) activity was measured using the SOD Assay Kit (Sigma-Aldrich). Protein arginine deiminase 4 (PAD4) expression was assessed by western blotting. Hydrogen peroxide (H2O2) was added exogenously in order to restore NETosis. Results HD isolated neutrophils exhibit decreased NETosis compared with normal controls in response to both PMA in the NOX-dependent pathway and CI in the NOX-independent pathway, as measured by immunofluorescence and cfDNA quantification. In the NOX-dependent pathway SOD activity was found to be 14% decreased in HD patients, resulting in the accumulation of superoxide radicals and decreased production of H2O2. In the NOX-independent pathway, PAD4 expression was found to be significantly decreased as well. NET formation was restored in vitro in HD neutrophils by the addition of exogenous H2O2. Conclusion To date, impaired NETosis was described only in congenital conditions such as chronic granulomatous disease and myeloperoxidase deficiency. To our knowledge, our research is the first to describe an acquired defect in NETosis in end stage renal disease patients undergoing chronic hemodialysis. An intervention aimed to improve neutrophil function in these patients may reduce the morbidity and mortality due to infection-related disease.

L-selectin function is required for beta 2-integrin-mediated neutrophil adhesion at physiological shear rates in vivo

1992 ◽  
Vol 263 (4) ◽  
pp. H1034-H1044 ◽  
Author(s):  
U. H. Von Andrian ◽  
P. Hansell ◽  
J. D. Chambers ◽  
E. M. Berger ◽  
I. Torres Filho ◽  
...  
Keyword(s):  
Neutrophil Function ◽  
Leukotriene B4 ◽  
Human Neutrophils ◽  
Neutrophil Adhesion ◽  
Shear Rates ◽  
Sequence Of Events ◽  
Multistep Process ◽  
Beta 2

In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.

Protein tyrosine phosphorylation mediates TNF-induced endothelial-neutrophil adhesion in vitro

1998 ◽  
Vol 274 (2) ◽  
pp. H513-H519 ◽  
Author(s):  
Susan A. Kelly ◽  
Pascal J. Goldschmidt-Clermont ◽  
Emily E. Milliken ◽  
Toshiyuki Arai ◽  
Elise H. Smith ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Adhesion Molecules ◽  
Neutrophil Adhesion ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Tnf Α ◽  
Dose Dependent

Proinflammatory cytokines initiate the vascular inflammatory response via the upregulation of adhesion molecules on the luminal endothelial surface. We investigated directly the role of protein tyrosine phosphorylation in the upregulation of the endothelial adhesion molecules, intercellular adhesion molecule 1 (ICAM-1) and E-selectin, and the consequent adhesion of neutrophils, after tumor necrosis factor (TNF)-α-stimulation of human aortic endothelial cells in vitro. Time- and dose-dependent TNF-α-stimulated ICAM-1 and E-selectin upregulation and neutrophil adhesion each were suppressed by tyrosine kinase inhibitors, including genistein (200 μM), but not genistin, its isoflavone analog without tyrosine kinase inhibitory activity. Tyrphostin AG 126, a synthetic selective tyrosine kinase inhibitor, also suppressed ICAM-1 and E-selectin upregulation and neutrophil adhesion, each in a dose-dependent manner, whereas tyrphostin AG 1288 had no effect. Tyrosine phosphorylation of two proteins (85 and 145 kDa in the cytoskeleton fraction) found minutes after TNF-α-stimulation was also inhibited by genistein. These findings suggest that, in endothelial cells, TNF-α upregulates ICAM-1 and E-selectin expression and consequent neutrophil adhesion via protein tyrosine phosphorylation.

Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2350-2350
Author(s):  
Antonella Zucchetto ◽  
Dania Benedetti ◽  
Claudio Tripodo ◽  
Riccardo Bomben ◽  
Fleur Bossi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Lymphocytes ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Biopsies ◽  
Tnf Α ◽  
Significant Difference

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods: CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results: IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions: CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures: No relevant conflicts of interest to declare.

CD38 Expression Marks CLL Cells with Invasive Properties

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2422-2422
Author(s):  
Tiziana Vaisitti ◽  
Cinzia Bologna ◽  
Sara Serra ◽  
Giovanni D'Arena ◽  
Davide Rossi ◽  
...  
Keyword(s):  
Prognostic Marker ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Genetic Manipulation ◽  
Active Form ◽  
Gelatin Zymography ◽  
Integrin Subunit ◽  
Proliferating Cells ◽  
Neoplastic Cells

Abstract Abstract 2422 CLL is characterized by a dynamic balance between proliferating cells in the lymphoid organs and circulating cells resisting programmed cell death. Regulating this equilibrium entails complex interactions between tumor and host, modulated by a set of surface molecules expressed by the CLL cell according to environmental conditions. CD38 is a negative prognostic marker, involved in the homing process. We previously reported that CD38+ cells are significantly more sensitive to CXCL12, a critical chemokine for the recirculation of neoplastic cells, both in vitro and in vivo. Activation of CD38 by means of agonistic mAbs promotes chemotaxis, while block of the molecule impairs it. CD38 is also co-expressed with CD49d, the alpha4 integrin subunit and a further independent negative prognostic marker for CLL. The two molecules appear to be intertwined in a dynamic loop which involves CD31 (the CD38 ligand predominantly expressed by endothelial cells) and VCAM-1 (the CD49d ligand). Attention has now been focused on MMP-9, the main matrix metalloproteinase expressed by CLL cells, due to the relevance of extra-vasation in the homing process. The final aim is to clarify whether CD38 may represent a key player, taking part to all the main steps of CLL cells recirculation. Results obtained analyzing a large cohort of CLL patients indicate that i) CD38+ cases are characterized by a higher expression and activity of MMP-9, as measured by gelatin zymography. Moreover, ii) the analysis of CLL patients with a bimodal expression of CD38 indicate that the CD38+ fraction of the clone is the one expressing higher levels of MMP-9 compared to the negative one. A formal proof of the connection between CD38 and this gelatinase has been obtained using a lentiviral technique that allows genetic manipulation of CLL cells. iii) De novo expression of CD38 is followed by secretion of high amounts of the active form of MMP-9, suggesting that de novo CD38+ cells digest extracellular matrix more readily. Furthermore, iv) the engagement of CD38 by means of an agonistic antibody is followed by an increased MMP-9 activation, while blocking anti-CD38 mAbs are highly effective in the modulation of MMP-9 secretion by CLL cells. Finally, v) CD38 appears to co-localize with MMP-9 in the same membrane areas, as inferred by confocal microscopy analysis. Considered together, these information pinpoint CD38 as a connecting element between chemokines, adhesion molecules and matrix metalloproteinases. The finding of a physical proximity of all these molecules suggests that they form a large supramolecular complex, with the characteristics of the invadosome, a podosome of neoplastic cells that controls diffusion and metastasis. If confirmed, these results would link the ability to migrate and invade tissues with an inferior clinical outcome also in leukemia and not only in solid tumors. Disclosures: No relevant conflicts of interest to declare.

Quantification of Anti-Sickling Effect of Aes-103 in Sickle Cell Disease Using an in Vitro Microfluidic Assay

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2699-2699 ◽  
Author(s):  
E. Du ◽  
Laurel Mendelsohn ◽  
James S. Nichols ◽  
Ming Dao ◽  
Gregory J. Kato
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Dependent Manner ◽  
Cell Disease ◽  
Pdms Film ◽  
Patient Sample ◽  
Hydroxyurea Treatment ◽  
O2 Concentration

Abstract Background: Under hypoxic conditions, sickle hemoglobin (HbS) polymerizes, causing morphologic distortion (sickling) of red blood cells (RBCs) in sickle cell disease (SCD). Aes-103 (5-hydroxymethylfurfural, 5-HMF) can stabilize the R-state and increase the oxygen affinity of hemoglobin, inhibiting the intracellular polymerization of HbS. Using a microfluidics-based hypoxia assay, we were able to track sickling of individual cells and quantify the anti-sickling effect of Aes-103 at millimolar (mM) levels in blood from SCD patients on hydroxyurea treatment (on-HU) and not on hydroxyurea treatment (off-HU). Method: We have developed a microfluidic assay that utilizes a gas permeable polydimethylsiloxane (PDMS) film 150 µm in thickness, to create a severe hypoxia microenvironment in a 5 µm deep chamber to measure cell sickling in vitro at 37°C. The hypoxia condition was 5 minutes in total, consisting of an initial oxygen-rich stage (20% O2), a transient deoxygenating stage (O2 concentration decreased to 5% within 15 second), and a steady-stage stage (O2 concentration decreased further and maintained at 2% for the rest of time). Blood samples from 3 on-HU and 3 off-HU patients were incubated with Aes-103 at concentrations of 0.5, 1, 2, and 5 mM for one hour at 37 degrees C, washed with Phosphate Buffered Saline and suspended in RPMI-1640 containing 1% w/v Bovine Serum Albumin for in vitro testing. Sickle RBCs undergoing sickling typically form spiky edges and a dark coarse texture due to intracellular HbS polymerization visually enhanced by a bandpass filter (Fig. 1A). The anti-sickling effect of Aes-103 was then quantified by the maximum sickled fraction (fraction of all RBCs that were morphologically distorted) under the hypoxia condition. Results: In the absence of Aes-103, the sickled fractions varied from 34% to 73% (Mean ± SD: 54% ± 18%). With the presence of Aes-103, the mean sickled fraction decreased with drug concentration (Fig. 1B), which can be well fitted with linear regression (R2= 0.95). With 2 mM Aes-103 incubation, each patient sample showed a significant decrease in cell sickling from its baseline. Addition of Aes-103 at 5 mM concentration prevented majority of RBCs from sickling (sickled fraction ≤ 5%). The sickled fraction of one patient sample was nearly zero. The distribution of sickled fractions does not completely correlate with the patient's HU status in this limited sample size (Fig. 1C). We also observed that hypoxia-induced sickling at baseline showed an apparent bimodal distribution, although the slope of response to Aes-103 concentration was similar. Conclusions: Our microfluidic assay enabled a rapid, quantitative characterization of cell sickling in vitro within a few minutes and using a single drop of whole blood patient sample. We confirmed the anti-sickling efficacy of Aes-103 for both on-HU and off-HU patient samples in a dosage-dependent manner. This assay has potential as a biomarker for drug development and monitoring for in vivo effect of potential anti-sickling therapeutics. Figure 1. (A) Identification of cell sickling from a microscopic image (arrows indicate the sickled RBCs). (B) Sickled fraction as a function of Aes-103 concentration. (C) Variation in response among different on-HU and off-HU patient samples. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

FXIIa Differentially Regulates Thrombin Generation during Steady State and Vaso-Occlusive Crisis in Sickle Cell Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 162-162 ◽  
Author(s):  
Erica M Sparkenbaugh ◽  
Camille Faes ◽  
Denis Noubouossie ◽  
Daniel K. Kirchhofer ◽  
András Gruber ◽  
...  
Keyword(s):  
Steady State ◽  
Mouse Model ◽  
Vascular Permeability ◽  
Sickle Cell ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Liver And Kidney

Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation. Previously, we demonstrated that inhibition of tissue factor (TF) attenuates thrombin generation (measured by plasma levels of thrombin-antithrombin complexes [TAT]) in a mouse model of SCD during steady state. Furthermore, we showed that neither inhibition of FXIIa-dependent activation of FXI (using 14E11 antibody) nor FXI deficiency reduces thrombin generation (TG) in sickle mice. In contrast, genetic deficiency of FXII or kininogen (HK) reduced plasma TAT levels. These data suggest that during steady state, FXIIa contributes to TG in sickle mice via activation of the kallikrein/HK pathway, but not FXI. In the present study, we further investigated the mechanisms of HK-induced TG at steady state, and increased TG observed during vaso-occlusive crisis (VOC). All experiments were performed using 4-5 month old Townes SS (sickle) and AA (control) mice. Kallikrein cleaves HK into HK fragments (HKFs) and bradykinin (BK). First, we investigated whether a BK-mediated increase in vascular permeability contributes to TG by exposing perivascular TF. This hypothesis was disproved by data demonstrating no difference in vascular permeability (measured by the extravasation of Evans blue in the heart, lung, liver and kidney) between AA (n=8) and SS (n=10) mice. HKFs were shown to induce leukocyte TF expression in vitro via binding to CD11b/CD18 (Mac-1). Therefore, we investigated whether Mac-1 inhibition affects TG in SS mice. AA and SS mice were treated with an inhibitory anti Mac-1 (M1/70) or IgG control antibody on days 0, 3 and 6 (i.p. 1 mg/kg) and TG was analyzed 1 day after the last injection. In the control group, SS mice demonstrated higher plasma TAT levels compared to AA mice (8.1±1.6 vs 4.2±0.6 ng/mL, n=10-11, p<0.05), but inhibition of Mac-1 significantly reduced plasma TAT levels in SS mice (4.6±0.7 ng/mL, n=11, p<0.05). These data suggest that HK might contribute to TG during steady state via Mac-1-dependent induction of monocyte TF. The steady state of SCD is interspersed with acute periods of VOC. Clinical data demonstrate that compared to the steady state, plasma levels of cell free DNA (cfDNA), activation of the contact system, and TG are further enhanced during VOC. To determine the mechanism of increased TG during VOC, we used the previously characterized mouse model of TNFα -induced VOC. Townes AA and SS mice were injected with recombinant TNFα (2 µg/g body weight) or the same volume of PBS, and plasma was collected 5 hours later. TNFα not only dramatically increased plasma levels of cfDNA in SS mice (14.78 ± 1.64 vs 679 ± 300 ng/mL; p<0.01), but also further increased plasma TAT levels compared to those observed in PBS-treated SS mice (2.9 fold, p<0.001, n=8). Importantly, there was a significant positive correlation between cfDNA and TAT in SS mice (r2 =0.65, p<0.001). Since cfDNA can activate FXII, we determined whether FXIIa-dependent activation of FXI contributes to TG during VOC. AA and SS mice received 14E11 or IgG control (4 mg/kg) 30 minutes before TNFα (2 μg/g) or PBS injection, and plasma TAT was assessed 5 hours later. Strikingly, 14E11 attenuated the increased TAT level in TNFα-treated SS mice, to the level observed in SS mice injected with PBS and IgG (IgG/SS/PBS: 9 ng/mL ± 1.8 vs. IgG/SS/TNF: 18.9 ± 3.6, p<0.001; 14E11/SS/TNF: 9.86 ± 0.72, p<0.05 vs. IgG/SS/TNF). We also determined if TF activity is required for the increased TG observed during VOC. Interestingly, inhibition of TF with an inhibitory 1H1 antibody (25 or 75 mg/kg injected i.p. 1 or 18 hours prior to TNFα, respectively) had no effect on the increased TG observed in TNFα treated SS mice. In aggregate, our data suggest that during the steady state of SCD, FXII-dependent TG is not FXI-dependent, but instead is mediated by a pathway involving HK, Mac-1 integrin and leukocyte TF. Furthermore, we propose that during VOC the massive release of cfDNA results in FXIIa-dependent FXI activation and enhances TG independently of TF. This study provides mechanistic insight into the initiators of TG in SCD. Moreover, it implicates FXIIa as a potential therapeutic target to reduce the prothrombotic state in SCD, during both steady state and VOC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pathological Role of Halted Proteasome Machinery in Sickle Cell Disease

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 955-955
Author(s):  
Anren Song ◽  
d'Alessandro Angelo ◽  
Kaiqi Sun ◽  
Hong Liu ◽  
Zhangzhe Peng ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Erythrocyte Membrane ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Proteomic Profiling ◽  
Cell Disease ◽  
E3 Ligases ◽  
Ubiquitinated Proteins

Abstract Although proteasome machinery is a conserved cellular component to maintain their normal function, its function in erythrocyte under stress conditions is largely unknown, especially in sickle cell disease (SCD). To determine whether proteasome machinery is altered in SCD erythrocyte, we conducted western blot to detect total ubiquitinated proteins on the erythrocyte membrane in both mice and humans with or without SCD. We found that ubiquitinated proteins were significantly accumulated in SCD mice and humans compared to WT mice and normal controls, indicating that proteasome machinery is halted in SCD. Next, to determine which specific proteins are ubiquitinated and accumulated in SCD, we conducted robust and nonbiased proteomic profiling by immunoprecipitation ubiquitinated proteins followed by proteomics analysis. We found significant accumulation of several categories of ubiquitinated proteins on the erythrocyte membrane in SCD, including cytoskeleton proteins (Spectrin, Actin, Ankryin), glycolytic enzymes (GAPDH, 2,3-BPG mutase, Pyruvate Kinase, G6PD), transporters (Band3, large neutral AA transporter, calcium transporter, ENT1), hemoglobin, components of proteasome machinery [E2, E3 ligases, and valosin-containing protein (p97)]. Finally, to determine the effect of halted proteasome machinery in SCD functionally, we conducted in vitro hypoxia induced red blood cell (RBC) sickling assay. We found that inhibition of RBC proteasome machinery by targeting p97 using CB-5083 or targeting proteasome using MG132 increases SCD RBC sickling. Overall, our findings reveal a novel role of halted proteasome machinery in the pathophysiology of SCD and open up new therapies for the disease. Disclosures No relevant conflicts of interest to declare.

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