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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 migration of PBL and T cell subsets through EC and their supporting Transwell filters. HUVEC were stimulated with 100 U/ml TNF alone, 10 ng/ml IFN alone, or both for 24 h. Lymphocytes were allowed to adhere and migrate for 24 h, after which, the number of lymphocytes transmigrating was counted and expressed as a percentage of the added cells. Data are mean ± sem from two to eight independent experiments.
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Figure 1: Effects of different cytokine treatments on migration of PBL and T cell subsets through EC and their supporting Transwell filters. HUVEC were stimulated with 100 U/ml TNF alone, 10 ng/ml IFN alone, or both for 24 h. Lymphocytes were allowed to adhere and migrate for 24 h, after which, the number of lymphocytes transmigrating was counted and expressed as a percentage of the added cells. Data are mean ± sem from two to eight independent experiments.

Mentions: Settling of lymphocytes onto EC filter constructs and quantification of the number collected from the back have been widely used to assess “transendothelial” migration. In initial experiments with unstimulated or TNF-treated HUVEC, we found that few PBL migrated through the filter after 2 h or 4 h (e.g., 2.4±1.0% of added cells migrated through TNF-stimulated HUVEC at 4 h; mean±sem; n=3). The proportion increased by 24 h (e.g., 11.0±3.2% of added cells that migrated through TNF-stimulated HUVEC; mean±sem; n=4), and so, we made comparisons between variously treated HUVEC at this time. Figure 1 shows that EC treated with cytokines (TNF or IFN alone or together) tended to support greater lymphocyte transmigration compared with unstimulated HUVEC, although there was no consistent difference between the cytokine treatments. This trend was also evident when CD4+ or CD8+ T cells were analyzed separately (Fig. 1), and the two types of T cells behaved similarly to each other. The proportion of lymphocytes that was adherent was high after 24 h (∼50%) and not affected significantly by cytokine treatments (data not shown). Such long contact times are not physiological and presumably increase nonspecific background adhesion. We thus reduced the initial contact time by washing off nonadherent lymphocytes after 10 min and maintained the 24-h migration endpoint. This decreased lymphocyte adhesion, and there was now a tendency toward greater adhesion for EC stimulated with TNF + IFN compared with untreated cells (21.8±3.7% vs. 14.8±6.0% of added PBL adherent, respectively; mean±SEM from six experiments). However, transmigration was also much lower (3.4±0.6% vs. 1.6±0.5% of added PBL transmigrated, respectively).


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 migration of PBL and T cell subsets through EC and their supporting Transwell filters. HUVEC were stimulated with 100 U/ml TNF alone, 10 ng/ml IFN alone, or both for 24 h. Lymphocytes were allowed to adhere and migrate for 24 h, after which, the number of lymphocytes transmigrating was counted and expressed as a percentage of the added cells. Data are mean ± sem from two to eight independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Effects of different cytokine treatments on migration of PBL and T cell subsets through EC and their supporting Transwell filters. HUVEC were stimulated with 100 U/ml TNF alone, 10 ng/ml IFN alone, or both for 24 h. Lymphocytes were allowed to adhere and migrate for 24 h, after which, the number of lymphocytes transmigrating was counted and expressed as a percentage of the added cells. Data are mean ± sem from two to eight independent experiments.
Mentions: Settling of lymphocytes onto EC filter constructs and quantification of the number collected from the back have been widely used to assess “transendothelial” migration. In initial experiments with unstimulated or TNF-treated HUVEC, we found that few PBL migrated through the filter after 2 h or 4 h (e.g., 2.4±1.0% of added cells migrated through TNF-stimulated HUVEC at 4 h; mean±sem; n=3). The proportion increased by 24 h (e.g., 11.0±3.2% of added cells that migrated through TNF-stimulated HUVEC; mean±sem; n=4), and so, we made comparisons between variously treated HUVEC at this time. Figure 1 shows that EC treated with cytokines (TNF or IFN alone or together) tended to support greater lymphocyte transmigration compared with unstimulated HUVEC, although there was no consistent difference between the cytokine treatments. This trend was also evident when CD4+ or CD8+ T cells were analyzed separately (Fig. 1), and the two types of T cells behaved similarly to each other. The proportion of lymphocytes that was adherent was high after 24 h (∼50%) and not affected significantly by cytokine treatments (data not shown). Such long contact times are not physiological and presumably increase nonspecific background adhesion. We thus reduced the initial contact time by washing off nonadherent lymphocytes after 10 min and maintained the 24-h migration endpoint. This decreased lymphocyte adhesion, and there was now a tendency toward greater adhesion for EC stimulated with TNF + IFN compared with untreated cells (21.8±3.7% vs. 14.8±6.0% of added PBL adherent, respectively; mean±SEM from six experiments). However, transmigration was also much lower (3.4±0.6% vs. 1.6±0.5% of added PBL transmigrated, respectively).

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