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Direct observations of the kinetics of migrating T cells suggest active retention by endothelial cells with continual bidirectional migration.

McGettrick HM, Hunter K, Moss PA, Buckley CD, Rainger GE, Nash GB - J. Leukoc. Biol. (2008)

Bottom Line: Under flow, migration kinetics and the proportions migrating back and forth were altered little.On collagen gels, PBL again crossed EC in minutes and migrated back and forth but showed little penetration of the gel over hours.In contrast, neutrophils migrated efficiently through EC and into gels.

View Article: PubMed Central - PubMed

Affiliation: The Medical School, The University of Birmingham, Birmingham B15 2TT, UK.

ABSTRACT
The kinetics and regulatory mechanisms of T cell migration through the endothelium have not been fully defined. In experimental, filter-based assays in vitro, transmigration of lymphocytes takes hours, compared with minutes, in vivo. We cultured endothelial cell (EC) monolayers on filters, solid substrates, or collagen gels and treated them with TNF-alpha, IFN-gamma, or both prior to analysis of lymphocyte migration in the presence or absence of flow. PBL, CD4+ cells, or CD8+ cells took many hours to migrate through EC-filter constructs for all cytokine treatments. However, direct microscopic observations of EC filters, which had been mounted in a flow chamber, showed that PBL crossed the endothelial monolayer in minutes and were highly motile in the subendothelial space. Migration through EC was also observed on clear plastic, with or without flow. After a brief settling without flow, PBL and isolated CD3+ or CD4+ cells crossed EC in minutes, but the numbers of migrated cells varied little with time. Close observation revealed that lymphocytes migrated back and forth continuously across endothelium. Under flow, migration kinetics and the proportions migrating back and forth were altered little. On collagen gels, PBL again crossed EC in minutes and migrated back and forth but showed little penetration of the gel over hours. In contrast, neutrophils migrated efficiently through EC and into gels. These observations suggest a novel model for lymphoid migration in which EC support migration but retain lymphocytes (as opposed to neutrophils), and additional signal(s) are required for onward migration.

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The migration behavior of individual lymphocytes over a 6-min period for HUVEC treated with TNF + IFN. (A) Video micrographs of a lymphocyte migrating from above the endothelial monolayer (phase-bright) to underneath (phase-dark), back to the top, and then under again. (B) Schematic representation of typical lymphocyte migratory behaviors. Individual cells were tracked over a 6-min period. Positions above (a) or below (b) the HUVEC monolayer are plotted for five cells: Cell 1 stayed above the monolayer, and Cell 5 stayed below. The others made one or more transits between the compartments. Similar behaviors were seen for HUVEC treated with TNF or IFN separately.
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Figure 4: The migration behavior of individual lymphocytes over a 6-min period for HUVEC treated with TNF + IFN. (A) Video micrographs of a lymphocyte migrating from above the endothelial monolayer (phase-bright) to underneath (phase-dark), back to the top, and then under again. (B) Schematic representation of typical lymphocyte migratory behaviors. Individual cells were tracked over a 6-min period. Positions above (a) or below (b) the HUVEC monolayer are plotted for five cells: Cell 1 stayed above the monolayer, and Cell 5 stayed below. The others made one or more transits between the compartments. Similar behaviors were seen for HUVEC treated with TNF or IFN separately.

Mentions: The detailed analysis of individual fields revealed another unexpected phenomenon. PBL were seen to continue transmigrating (going from phase-bright to phase-dark) throughout the entire 15-min period, but other cells were migrating in the opposite (basal-to-apical) direction. The continual forward and reverse migration explained the nearly constant level of transmigration observed at any time. Some cells made several transits back and forth within the observation period, and some stayed in the same compartment throughout. To quantify this behavior, we followed individual cells second-by-second over a period of 6 min and recorded if and when they moved between the basal and apical surfaces of the HUVEC. A sequence of pictures of a multiply migrating cell is shown in Figure 4A, and some typical behaviors are illustrated schematically in Figure 4B. For cytokine-treated HUVEC, ∼20% of PBL made at least one transit during the observation period, and about one-third of these made multiple transits (Table 1A). By measuring the number of transits during the 6-min period, we calculated the average interval between transits for those cells that moved between compartments (mean ∼4 min; Table 1A). The values varied little between the cytokine treatments (TNF, IFN, or TNF+IFN). Transits were seen with unstimulated HUVEC (data not shown), but again, the number of cells observed was small. On average, half of the transits were forward and half in the reverse direction, which was consistent with the observation noted above—that overall levels of transmigration were constant over the total observation period.


Direct observations of the kinetics of migrating T cells suggest active retention by endothelial cells with continual bidirectional migration.

McGettrick HM, Hunter K, Moss PA, Buckley CD, Rainger GE, Nash GB - J. Leukoc. Biol. (2008)

The migration behavior of individual lymphocytes over a 6-min period for HUVEC treated with TNF + IFN. (A) Video micrographs of a lymphocyte migrating from above the endothelial monolayer (phase-bright) to underneath (phase-dark), back to the top, and then under again. (B) Schematic representation of typical lymphocyte migratory behaviors. Individual cells were tracked over a 6-min period. Positions above (a) or below (b) the HUVEC monolayer are plotted for five cells: Cell 1 stayed above the monolayer, and Cell 5 stayed below. The others made one or more transits between the compartments. Similar behaviors were seen for HUVEC treated with TNF or IFN separately.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2626767&req=5

Figure 4: The migration behavior of individual lymphocytes over a 6-min period for HUVEC treated with TNF + IFN. (A) Video micrographs of a lymphocyte migrating from above the endothelial monolayer (phase-bright) to underneath (phase-dark), back to the top, and then under again. (B) Schematic representation of typical lymphocyte migratory behaviors. Individual cells were tracked over a 6-min period. Positions above (a) or below (b) the HUVEC monolayer are plotted for five cells: Cell 1 stayed above the monolayer, and Cell 5 stayed below. The others made one or more transits between the compartments. Similar behaviors were seen for HUVEC treated with TNF or IFN separately.
Mentions: The detailed analysis of individual fields revealed another unexpected phenomenon. PBL were seen to continue transmigrating (going from phase-bright to phase-dark) throughout the entire 15-min period, but other cells were migrating in the opposite (basal-to-apical) direction. The continual forward and reverse migration explained the nearly constant level of transmigration observed at any time. Some cells made several transits back and forth within the observation period, and some stayed in the same compartment throughout. To quantify this behavior, we followed individual cells second-by-second over a period of 6 min and recorded if and when they moved between the basal and apical surfaces of the HUVEC. A sequence of pictures of a multiply migrating cell is shown in Figure 4A, and some typical behaviors are illustrated schematically in Figure 4B. For cytokine-treated HUVEC, ∼20% of PBL made at least one transit during the observation period, and about one-third of these made multiple transits (Table 1A). By measuring the number of transits during the 6-min period, we calculated the average interval between transits for those cells that moved between compartments (mean ∼4 min; Table 1A). The values varied little between the cytokine treatments (TNF, IFN, or TNF+IFN). Transits were seen with unstimulated HUVEC (data not shown), but again, the number of cells observed was small. On average, half of the transits were forward and half in the reverse direction, which was consistent with the observation noted above—that overall levels of transmigration were constant over the total observation period.

Bottom Line: Under flow, migration kinetics and the proportions migrating back and forth were altered little.On collagen gels, PBL again crossed EC in minutes and migrated back and forth but showed little penetration of the gel over hours.In contrast, neutrophils migrated efficiently through EC and into gels.

View Article: PubMed Central - PubMed

Affiliation: The Medical School, The University of Birmingham, Birmingham B15 2TT, UK.

ABSTRACT
The kinetics and regulatory mechanisms of T cell migration through the endothelium have not been fully defined. In experimental, filter-based assays in vitro, transmigration of lymphocytes takes hours, compared with minutes, in vivo. We cultured endothelial cell (EC) monolayers on filters, solid substrates, or collagen gels and treated them with TNF-alpha, IFN-gamma, or both prior to analysis of lymphocyte migration in the presence or absence of flow. PBL, CD4+ cells, or CD8+ cells took many hours to migrate through EC-filter constructs for all cytokine treatments. However, direct microscopic observations of EC filters, which had been mounted in a flow chamber, showed that PBL crossed the endothelial monolayer in minutes and were highly motile in the subendothelial space. Migration through EC was also observed on clear plastic, with or without flow. After a brief settling without flow, PBL and isolated CD3+ or CD4+ cells crossed EC in minutes, but the numbers of migrated cells varied little with time. Close observation revealed that lymphocytes migrated back and forth continuously across endothelium. Under flow, migration kinetics and the proportions migrating back and forth were altered little. On collagen gels, PBL again crossed EC in minutes and migrated back and forth but showed little penetration of the gel over hours. In contrast, neutrophils migrated efficiently through EC and into gels. These observations suggest a novel model for lymphoid migration in which EC support migration but retain lymphocytes (as opposed to neutrophils), and additional signal(s) are required for onward migration.

Show MeSH
Related in: MedlinePlus