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.

scholarly journals BIOPHYSICAL PROPERTIES OF CELL SURFACE AND FUNCTIONAL ACTIVITY OF GRANULOCYTES OF PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA DURING MODELING OF ATP LOAD IN VITRO

2021 ◽  
Vol 10 (3) ◽  
pp. 43-47
Author(s):  
M. Yu. Skorkina ◽  
N. I. Zhernakova ◽  
T. S. Shevchenko ◽  
A. S. Zelentsova
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Surface ◽  
Functional Activity ◽  
Lymphoblastic Leukemia ◽  
Membrane Surface ◽  
Disodium Salt ◽  
Biophysical Properties ◽  
Adhesive Properties ◽  
Migration Activity

The current study is devoted to the investigation of the biophysical properties of the cell surface and the functional activity of granulocytes in patients with acute lymphoblastic leukemia when simulating the ATP load in vitro. The experiment was performed on the peripheral blood of patients with acute lymphoblastic leukemia (ALL) who underwent a standard course of chemotherapy. In experimental tests, exogenous loading with ATP was simulated in vitro by adding 100.0 μM adenosine-5-triphosphate disodium salt trihydrate to the granulocyte suspension. Incubation with the drug was carried out for 15 min at 370 C. As a control, a suspension of granulocytes in RPMI 1640 medium from the same patient, but without the addition of the drug, was used, this was incubated for 15 min at a temperature of 370 C. After the incubation time, the biophysical properties (rigidity, charge of the cell surface, the strength of intercellular adhesion) of granulocytes in experimental and control samples were studied using atomic force microscopy, and the migration activity of cells was also assessed in a direct capillary test under agarose. A model with exogenous ATP in ALL patients showed a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties and migration activity of granulocytes. The revealed effects point to the key role of the ATP molecule in the mechanisms of intercellular signaling in the microvasculature.

The nature of leukocyte shape changes in the pulmonary capillaries

1997 ◽  
Vol 273 (4) ◽  
pp. L733-L740 ◽  
Author(s):  
Darlene M. Redenbach ◽  
Dean English ◽  
James C. Hogg
Keyword(s):  
Blood Cells ◽  
White Blood Cells ◽  
Cell Polarization ◽  
Cell Counts ◽  
Pulmonary Capillaries ◽  
Size Discrepancy ◽  
Capillary Bed ◽  
Washout Curves ◽  
Shape Changes

The size discrepancy between leukocytes [white blood cells (WBCs)] and pulmonary capillaries requires WBCs to deform. We investigated the persistence of this deformation on cells leaving the capillary bed and the role played by the cytoskeleton. Isolated rabbit lungs were perfused in situ via the pulmonary artery with effluent fractions collected from the left ventricle. Washout curves from cell counts in each fraction confirmed that WBCs are preferentially retained over erythrocytes. WBC deformation present on exit from the circulation was compared with that present after recovery in paired fractions, fixed either immediately or 60 min later. These cells were compared with cells recovered from the capillary in perfused fixative or fixed in peripheral blood. Our results show that leukocyte deformation persisted after the cells exited the pulmonary circulation. This deformation was associated with minimal submembranous F-actin staining, and microtubule distribution and cell polarization were unchanged. We conclude that cytoskeletal changes that occur during WBC deformation in the pulmonary capillaries are minimal and differ from those known to occur in actively migrating cells during chemotaxis.

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.

scholarly journals Sex-dependent dysregulation of human neutrophil responses by bisphenol A

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Marzena Garley ◽  
Malgorzata Rusak ◽  
Karolina Nowak ◽  
Jan Czerniecki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Bisphenol A ◽  
Total Concentration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
No Production ◽  
Pathogen Elimination ◽  
Latex Beads

Abstract Background In the present study, we aimed to investigate selected functions of human neutrophils exposed to bisphenol A (BPA) under in vitro conditions. As BPA is classified among xenoestrogens, we compared its action and effects with those of 17β-estradiol (E2). Methods Chemotaxis of neutrophils was examined using the Boyden chamber. Their phagocytosis and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity were assessed via Park’s method with latex beads and Park’s test with nitroblue tetrazolium. To assess the total concentration of nitric oxide (NO), the Griess reaction was utilized. Flow cytometry was used to assess the expression of cluster of differentiation (CD) antigens. The formation of neutrophil extracellular traps (NETs) was analyzed using a microscope (IN Cell Analyzer 2200 system). Expression of the investigated proteins was determined using Western blot. Results The analysis of results obtained for both sexes demonstrated that after exposure to BPA, the chemotactic capacity of neutrophils was reduced. In the presence of BPA, the phagocytic activity was found to be elevated in the cells obtained from women and reduced in the cells from men. Following exposure to BPA, the percentage of neutrophils with CD14 and CD284 (TLR4) expression, as well as the percentage of cells forming NETs, was increased in the cells from both sexes. The stimulatory role of BPA and E2 in the activation of NADPH oxidase was observed only in female cells. On the other hand, no influence of E2 on the expression of CD14 and CD284, chemotaxis, phagocytosis, and the amount of NET-positive neutrophils was found for both sexes. The study further showed that BPA intensified NO production and iNOS expression in the cells of both sexes. In addition, intensified expression of all tested PI3K-Akt pathway proteins was observed in male neutrophils. Conclusions The study demonstrated the influence of BPA on neutrophil functions associated with locomotion and pathogen elimination, which in turn may disturb the immune response of these cells in both women and men. Analysis of the obtained data showed that the effect of this xenoestrogen on the human neutrophils was more pronounced than E2.

scholarly journals Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
M. Adamczyk ◽  
E. Lewicka ◽  
R. Szatkowska ◽  
H. Nieznanska ◽  
J. Ludwiczak ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Range ◽  
Coiled Coil ◽  
Amino Acid Sequences ◽  
Broad Host Range ◽  
Biophysical Properties ◽  
Dna Binding Sites ◽  
In Silico Studies ◽  
Wide Range

Abstract Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

scholarly journals Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2005
Author(s):  
Irene Vorontsova ◽  
James E. Hall ◽  
Thomas F. Schilling ◽  
Noriaki Nagai ◽  
Yosuke Nakazawa
Keyword(s):  
Cell Adhesion ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Adhesion Assay ◽  
Loss Of Function ◽  
Adhesive Properties ◽  
The Difference ◽  
In Vitro Adhesion ◽  
Cell To Cell Adhesion

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

scholarly journals Vascularized organoids on a chip: strategies for engineering organoids with functional vasculature

Lab on a Chip ◽  
2021 ◽  
Vol 21 (3) ◽  
pp. 473-488
Author(s):  
Shun Zhang ◽  
Zhengpeng Wan ◽  
Roger D. Kamm
Keyword(s):  
Capillary Bed

Possible strategy to integrate pre-vascularized organoid and in vitro capillary bed on a microfluidic based platform, aiming for establishing perfused vasculature throughout organoids in vitro.

scholarly journals A sand fly salivary protein acts as a neutrophil chemoattractant

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anderson B. Guimaraes-Costa ◽  
John P. Shannon ◽  
Ingrid Waclawiak ◽  
Jullyanna Oliveira ◽  
Claudio Meneses ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Calcium Influx ◽  
Parasite Burden ◽  
Salivary Protein ◽  
Sand Fly ◽  
Human Neutrophils ◽  
Chemotactic Protein ◽  
The Impact

AbstractApart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

scholarly journals Intermittent Hypoxia Affects the Spontaneous DifferentiationIn Vitroof Human Neutrophils into Long-Lived Giant Phagocytes

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Larissa Dyugovskaya ◽  
Slava Berger ◽  
Andrey Polyakov ◽  
Peretz Lavie ◽  
Lena Lavie
Keyword(s):  
Nadph Oxidase ◽  
Intermittent Hypoxia ◽  
Oxidase Inhibitor ◽  
Human Neutrophils ◽  
Hypoxic Conditions ◽  
Oxygen Conditions ◽  
Extended Lifespan ◽  
First Time ◽  
Dose Dependent

Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (Gϕ) which spontaneously developin vitrowithout additional growth factors or cytokines. Gϕare CD66b+/CD63+/MPO+/LC3B+and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS) production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕformation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH) (29 cycles/day for 5 days) completely abolished Gϕformation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles) decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phoxexpression, whereas under 24 h sustained hypoxia (SH) the size and expression of LC3B and gp91-phox/p22-phoxresembled Gϕformed in normoxia. Diphenyl iodide (DPI), a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕformation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC) abrogated the effects of IH by inducing large CD66b+/LC3B+Gϕand increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕdevelopment.

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