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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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Effects of different cytokine treatments on recruitment of lymphocytes to EC in a flow-based assay. A 4-min bolus of lymphocytes was perfused over HUVEC that had been cultured on chamber slides and treated with TNF alone, IFN alone, or both for 24 h. (A) Effects of cytokines on lymphocyte adhesion measured at 2 min after wash-off. (B) The behavior of the adherent lymphocytes [rolling, stationary-adherent, or migrated through the endothelial monolayer, transendothelial migration (TEM)] measured 11 min after wash-off. Data are mean ± sem from three to four independent experiments. (A) ANOVA showed a significant effect of treatment (P<0.01). *, P < 0.05; **, P < 0.01, compared with untreated by Dunnett test.
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Figure 6: Effects of different cytokine treatments on recruitment of lymphocytes to EC in a flow-based assay. A 4-min bolus of lymphocytes was perfused over HUVEC that had been cultured on chamber slides and treated with TNF alone, IFN alone, or both for 24 h. (A) Effects of cytokines on lymphocyte adhesion measured at 2 min after wash-off. (B) The behavior of the adherent lymphocytes [rolling, stationary-adherent, or migrated through the endothelial monolayer, transendothelial migration (TEM)] measured 11 min after wash-off. Data are mean ± sem from three to four independent experiments. (A) ANOVA showed a significant effect of treatment (P<0.01). *, P < 0.05; **, P < 0.01, compared with untreated by Dunnett test.

Mentions: Unstimulated EC cultured in chamber slides consistently failed to recruit flowing lymphocytes (Fig. 6A). However, after cytokine stimulation, the EC efficiently captured much greater numbers of flowing lymphocytes, with greater adhesion observed on EC stimulated with TNF or TNF + IFN compared with IFN alone (Fig. 6A). As with HUVEC cultured on filters, few captured lymphocytes rolled, and most adhered firmly. Nearly one-half of the adherent PBL transmigrated through the cytokine-stimulated monolayers within 11 min, and IFN was the most effective inducer of migration (Fig. 4B). Velocity of migrated cells averaged ∼8 μm/min, which is similar to that observed in the static assay, and again, velocity tended to be faster in the presence of IFN (Table 1C).


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)

Effects of different cytokine treatments on recruitment of lymphocytes to EC in a flow-based assay. A 4-min bolus of lymphocytes was perfused over HUVEC that had been cultured on chamber slides and treated with TNF alone, IFN alone, or both for 24 h. (A) Effects of cytokines on lymphocyte adhesion measured at 2 min after wash-off. (B) The behavior of the adherent lymphocytes [rolling, stationary-adherent, or migrated through the endothelial monolayer, transendothelial migration (TEM)] measured 11 min after wash-off. Data are mean ± sem from three to four independent experiments. (A) ANOVA showed a significant effect of treatment (P<0.01). *, P < 0.05; **, P < 0.01, compared with untreated by Dunnett test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effects of different cytokine treatments on recruitment of lymphocytes to EC in a flow-based assay. A 4-min bolus of lymphocytes was perfused over HUVEC that had been cultured on chamber slides and treated with TNF alone, IFN alone, or both for 24 h. (A) Effects of cytokines on lymphocyte adhesion measured at 2 min after wash-off. (B) The behavior of the adherent lymphocytes [rolling, stationary-adherent, or migrated through the endothelial monolayer, transendothelial migration (TEM)] measured 11 min after wash-off. Data are mean ± sem from three to four independent experiments. (A) ANOVA showed a significant effect of treatment (P<0.01). *, P < 0.05; **, P < 0.01, compared with untreated by Dunnett test.
Mentions: Unstimulated EC cultured in chamber slides consistently failed to recruit flowing lymphocytes (Fig. 6A). However, after cytokine stimulation, the EC efficiently captured much greater numbers of flowing lymphocytes, with greater adhesion observed on EC stimulated with TNF or TNF + IFN compared with IFN alone (Fig. 6A). As with HUVEC cultured on filters, few captured lymphocytes rolled, and most adhered firmly. Nearly one-half of the adherent PBL transmigrated through the cytokine-stimulated monolayers within 11 min, and IFN was the most effective inducer of migration (Fig. 4B). Velocity of migrated cells averaged ∼8 μm/min, which is similar to that observed in the static assay, and again, velocity tended to be faster in the presence of IFN (Table 1C).

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