scholarly journals The effect of neutrophil migration on epithelial permeability.

1986 ◽  
Vol 103 (6) ◽  
pp. 2729-2738 ◽  
Author(s):  
L C Milks ◽  
G P Conyers ◽  
E B Cramer
Keyword(s):  
Electrical Resistance ◽  
Neutrophil Migration ◽  
Inflammatory Lesion ◽  
Time Frame ◽  
Transepithelial Electrical Resistance ◽  
Human Neutrophils ◽  
Epithelial Permeability ◽  
Vitro Model ◽  
Migratory Cells

To reach an inflammatory lesion, neutrophils must frequently traverse the epithelium of an infected organ. Whether the actual migration of neutrophils alters the epithelial permeability is unknown. Through the use of an in vitro model system it was possible to directly determine the effect of neutrophil emigration on the transepithelial electrical resistance of the monolayer. Human neutrophils (5 X 10(6) cells/ml) were placed in the upper compartment of a combined chemotaxis/resistance chamber and stimulated for 40 min by a gradient of 10(-7) M n-formyl-methionyl-leucyl-phenylalanine to traverse a confluent monolayer of canine kidney epithelial cells grown on micropore filters. Neither the chemoattractant alone (10(-5)-10(-9) M) nor the accumulation of an average of eight neutrophils per millimeter of epithelium lowered the transepithelial electrical resistance. However, under certain conditions the migration of neutrophils temporarily increased the permeability of the monolayer. The resistance fell approximately 48% within 5 min if the migratory cells were stimulated to reverse their migration across the same monolayer. As re-migration continued, the resistance returned to its initial levels within 60 min. Doubling the initial neutrophil concentration to 10 X 10(6) cells/ml resulted in the accumulation of an average of 66 neutrophils per millimeter of epithelium and an average fall in resistance of 46% (r = 0.98; P less than 0.001) in 40 min. If the resistance had fallen less than 45%, removal of the neutrophils remaining in the upper compartment resulted in a return of the transepithelial electrical resistance to its initial level within 65 min. However, when the fall was greater than 45%, the resistance only recovered to 23.5% of its initial levels within the same time frame. Thus, these results suggest that the integrity of an epithelium can, under certain conditions, be affected by the emigration of neutrophils, but that this effect is either completely or partially reversible within 65 min.

scholarly journals Epithelial permeability and the transepithelial migration of human neutrophils.

1983 ◽  
Vol 96 (5) ◽  
pp. 1241-1247 ◽  
Author(s):  
L C Milks ◽  
M J Brontoli ◽  
E B Cramer
Keyword(s):  
In Vitro Model ◽  
The Body ◽  
Human Neutrophils ◽  
Epithelial Permeability ◽  
Cell Contacts ◽  
Epithelial Monolayers ◽  
Tissue Factors ◽  
Vitro Model ◽  
Close Contacts

Although polymorphonuclear leukocytes (PMN's) can migrate through every epithelium in the body regardless of its permeability, very little is known about the effect of epithelial permeability on PMN migration and the effect of emigrating PMN's on the permeability of the epithelium. In an in vitro model system of transepithelial migration, human PMN's were stimulated by 0.1 micrometer fMet-Leu-Phe to traverse confluent, polarized canine kidney epithelial monolayers of varying permeabilities. Epithelial permeability was determined by both conductance measurement and horseradish peroxidase (HRP) tracer studies. As epithelial permeability increased, the number of PMN invasion sites as well as the number of PMN's that traversed the monolayer increased. The effect of PMN migration on epithelial permeability was examined using the ultrastructural tracers HRP and lanthanum nitrate. PMN's traversing the monolayer made close cell-to-cell contacts with other invading PMNs and with adjacent epithelial cells. These close contacts appeared to prevent leakage of tracer across invasion sites. Following PMN emigration, epithelial junctional membranes reapproximated and were impermeable to the tracers. These results indicated that, in the absence of serum and connective tissue factors, (a) the number of PMN invasion sites and the number of PMN's that traversed an epithelium were a function of the conductance of the epithelium and (b) PMN's in the process of transepithelial migration maintained close cell-cell contacts and prevented the leakage of particles (greater than 5 nm in diameter) across the invasion site.

Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation

2008 ◽  
Vol 294 (4) ◽  
pp. G906-G913 ◽  
Author(s):  
Francesco Raimondi ◽  
Pasquale Santoro ◽  
Maria Vittoria Barone ◽  
Serena Pappacoda ◽  
Maria Luisa Barretta ◽  
...  
Keyword(s):  
Tight Junction ◽  
Bile Acids ◽  
Electrical Resistance ◽  
Egf Receptor ◽  
Time Frame ◽  
Paracellular Permeability ◽  
Transepithelial Electrical Resistance ◽  
Confocal Laser ◽  
Gut Permeability

Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 μM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 μM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.

LLC-PK1 cells: cloning of phenotypically stable subpopulations

1986 ◽  
Vol 250 (5) ◽  
pp. C682-C687 ◽  
Author(s):  
A. Wohlwend ◽  
J. D. Vassalli ◽  
D. Belin ◽  
L. Orci
Keyword(s):  
Cell Line ◽  
Plasminogen Activator ◽  
Electrical Resistance ◽  
In Vitro Model ◽  
Fold Increase ◽  
Transepithelial Electrical Resistance ◽  
Pig Kidney ◽  
Vitro Model ◽  
Original Cell

The LLC-PK1 pig kidney-derived cell line is morphologically and functionally heterogeneous. We have clonally derived three sublines that differ in their response to calcitonin and in their ability to form domes. The three clones were analyzed for their basal and hormonally induced plasminogen activator production. In contrast to the D + Sc clone, in which calcitonin induced a greater than 100-fold increase in plasminogen activator synthesis, the D + Rc clone did not respond to the hormone; this was related to a deficiency of the cells in calcitonin binding. Transepithelial electrical resistance measurements revealed a direct correlation with the capacity of the cells to form domes; in one of the isolated clones (D-), the lack of dome formation coincided with a low electrical resistance; the D + Sc clone, in which all single cell-derived colonies formed domes, showed a higher electrical resistance than that developed by the original cell line. Thus the LLC-PK1 clones provide a useful in vitro model for the study of epithelial properties.

Transepithelial Resistance and Inulin Permeability as Endpoints in In Vitro Nephrotoxicity Testing

2002 ◽  
Vol 30 (2_suppl) ◽  
pp. 53-59 ◽  
Author(s):  
Tracey Duff ◽  
Simon Carter ◽  
Gemma Feldman ◽  
Gordon McEwan ◽  
Walter Pfaller ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Electrical Resistance ◽  
Mdck Cells ◽  
Ussing Chamber ◽  
Fluorescein Isothiocyanate ◽  
Transepithelial Electrical Resistance ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Transepithelial electrical resistance (RT) and the flux of fluorescein isothiocyanate (FITC) across Madin Darby canine kidney (MDCK) strain 1 cells and porcine epithelial kidney (LLC-PK1) monolayers were compared between three laboratories for a range of nephrotoxins. The precision of the REMS AutoSampler was similar to that of the Ussing chamber and the ENDOHM® technique, but superior to using chopstick electrodes, for measurements of resistance. The nephrotoxins used were selective for the proximal tubule, and in all cases, LLC-PK1 cells were more sensitive than MDCK cells. In most cases, change in RT was a more sensitive indicator of damage than alterations in FITC flux. The REMS system provides high intra-plate precision for RT measurements and is a higher throughput system, which is applicable to screening for nephrotoxicity in vitro.

Effects of Fermentation Products of Pro- and Prebiotics on Trans-Epithelial Electrical Resistance in an In Vitro Model of the Colon

2005 ◽  
Vol 51 (1) ◽  
pp. 102-109 ◽  
Author(s):  
Daniel M. Commane ◽  
Colette T. Shortt ◽  
Stefania Silvi ◽  
Albert Cresci ◽  
Roisin M. Hughes ◽  
...  
Keyword(s):  
Electrical Resistance ◽  
In Vitro Model ◽  
Fermentation Products ◽  
Vitro Model

A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

2019 ◽  
Vol 44 (6) ◽  
pp. 810-821
Author(s):  
Edibe Avci ◽  
Yeliz Z. Akkaya-Ulum ◽  
Digdem Yoyen-Ermis ◽  
Gunes Esendagli ◽  
Banu Balci-Peynircioglu
Keyword(s):  
Flow Cytometry ◽  
Cell Migration ◽  
Neutrophil Migration ◽  
Cost Effective ◽  
In Vitro Assays ◽  
Human Neutrophils ◽  
Neutrophil Granulocytes ◽  
Inflammatory Conditions ◽  
Migration Analysis

Abstract Background Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders.

Filter-grown TR146 cells as an in vitro model of human buccal epithelial permeability

1999 ◽  
Vol 107 (2) ◽  
pp. 138-146 ◽  
Author(s):  
Jette Jacobsen ◽  
Eva Bonde Nielsen ◽  
Karen Brøndum-Nielsen ◽  
Maria Elisabeth Christensen ◽  
Helle-Birgitte Dahl Olin ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Epithelial Permeability ◽  
Vitro Model

Leukocyte antibacterial functions are not impaired by perfluorocarbon exposure in vitro

2008 ◽  
Vol 295 (1) ◽  
pp. L134-L142 ◽  
Author(s):  
Dirk Haufe ◽  
Eva Koenigshausen ◽  
Lilla Knels ◽  
Martina Wendel ◽  
Sebastian N. Stehr ◽  
...  
Keyword(s):  
Host Defense ◽  
In Vitro Model ◽  
Polymorphonuclear Neutrophils ◽  
Human Neutrophils ◽  
Intracellular Uptake ◽  
E Coli ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Vitro Model

Application of liquid, aerosolized, and vaporized perfluorocarbons (PFC) in acute lung injury has shown anti-inflammatory effects. Although this may be beneficial in states of pulmonary hyperinflammation, it also could increase susceptibility to nosocomial lung infection. We hypothesized that PFC impair cellular host defense and therefore investigated in an in vitro model the influence of perfluorohexane (PFH) on crucial mechanisms of bacterial elimination in human neutrophils and monocytes. Using scanning and transmission electron microscopy, we could show membrane-bound and ingested PFH particles that morphologically did not alter adherence and phagocytosis of Escherichia coli or leukocyte viability. The amount of adherent and phagocytosed bacteria as determined by flow cytometry was not influenced in cells only pretreated with PFH for 1 and 4 h. When PFH was present during E. coli challenge, bacterial adherence was decreased in polymorphonuclear neutrophils, but respective intracellular uptake was not impaired and was even significantly promoted in monocytes. Overall, E. coli-induced respiratory burst capacity was not reduced by PFH. Our findings provide evidence that key functions of innate host defense are not compromised by PFH treatment in vitro.

scholarly journals Effect of human serum and some of its components on neutrophil adherence and migration across an epithelium.

1986 ◽  
Vol 102 (5) ◽  
pp. 1868-1877 ◽  
Author(s):  
E B Cramer ◽  
L C Milks ◽  
M J Brontoli ◽  
G K Ojakian ◽  
S D Wright ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Serum ◽  
Neutrophil Migration ◽  
Autologous Serum ◽  
Heat Inactivation ◽  
Human Neutrophils ◽  
Epithelial Surface ◽  
Neutrophil Adherence ◽  
And Migration

The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.

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