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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 lymphocyte recruitment to EC cultured in multiwell plates. HUVEC were stimulated with 100 U/ml TNF alone, 10 ng/ml IFN alone, or both for 24 h. Lymphocytes were allowed to settle for 5 min, nonadherent cells were washed off, and lymphocyte adhesion and transmigration were analyzed by phase-contrast microscopy. (A) Effect of cytokines on lymphocyte adhesion measured at 2 min after wash-off. (B) Effect of cytokines on lymphocyte transmigration through HUVEC at 2 min and 17 min after wash-off. (C) Time courses of lymphocyte transendothelial migration for different cytokines. Data are mean ± sem from four independent experiments. (A and B) ANOVA showed a significant effect of cytokine treatment on lymphocyte adhesion and transmigration; P < 0.01; *, P < 0.05; **, P < 0.01, compared with untreated by Dunnett test.
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Figure 3: Effects of different cytokine treatments on lymphocyte recruitment to EC cultured in multiwell plates. HUVEC were stimulated with 100 U/ml TNF alone, 10 ng/ml IFN alone, or both for 24 h. Lymphocytes were allowed to settle for 5 min, nonadherent cells were washed off, and lymphocyte adhesion and transmigration were analyzed by phase-contrast microscopy. (A) Effect of cytokines on lymphocyte adhesion measured at 2 min after wash-off. (B) Effect of cytokines on lymphocyte transmigration through HUVEC at 2 min and 17 min after wash-off. (C) Time courses of lymphocyte transendothelial migration for different cytokines. Data are mean ± sem from four independent experiments. (A and B) ANOVA showed a significant effect of cytokine treatment on lymphocyte adhesion and transmigration; P < 0.01; *, P < 0.05; **, P < 0.01, compared with untreated by Dunnett test.

Mentions: When PBL were allowed to settle for 5 min, few (∼5%) adhered to unstimulated HUVEC cultured in multiwell plates, but cytokine-stimulated EC supported much higher levels of attachment (Fig. 3A). We were surprised to find that 2 min after washing, a significant proportion of the adherent cells had transmigrated and that after a further 15 min, this proportion remained essentially the same (Fig. 3B). Transmigration was higher for cytokine-treated monolayers, particularly in the presence of IFN. Although some adherent cells did transmigrate through unstimulated HUVEC in minutes, the absolute number observed was small as a result of the low level of adhesion. Examining migration through cytokine-treated EC in more detail, we recorded individual fields and repeatedly assessed transmigration. Although there were minor fluctuations in the proportion of PBL transmigrated, there were no significant upward trends (Fig. 3C). We also noted the velocity of migrated cells, which averaged ∼8 μm/min and tended to be faster in the presence of IFN (Table 1A). Thus, in a static assay with short initial contact times, we detected cytokine-specific induction of lymphocyte adhesion and could observe transendothelial migration within minutes.


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 lymphocyte recruitment to EC cultured in multiwell plates. HUVEC were stimulated with 100 U/ml TNF alone, 10 ng/ml IFN alone, or both for 24 h. Lymphocytes were allowed to settle for 5 min, nonadherent cells were washed off, and lymphocyte adhesion and transmigration were analyzed by phase-contrast microscopy. (A) Effect of cytokines on lymphocyte adhesion measured at 2 min after wash-off. (B) Effect of cytokines on lymphocyte transmigration through HUVEC at 2 min and 17 min after wash-off. (C) Time courses of lymphocyte transendothelial migration for different cytokines. Data are mean ± sem from four independent experiments. (A and B) ANOVA showed a significant effect of cytokine treatment on lymphocyte adhesion and transmigration; 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 3: Effects of different cytokine treatments on lymphocyte recruitment to EC cultured in multiwell plates. HUVEC were stimulated with 100 U/ml TNF alone, 10 ng/ml IFN alone, or both for 24 h. Lymphocytes were allowed to settle for 5 min, nonadherent cells were washed off, and lymphocyte adhesion and transmigration were analyzed by phase-contrast microscopy. (A) Effect of cytokines on lymphocyte adhesion measured at 2 min after wash-off. (B) Effect of cytokines on lymphocyte transmigration through HUVEC at 2 min and 17 min after wash-off. (C) Time courses of lymphocyte transendothelial migration for different cytokines. Data are mean ± sem from four independent experiments. (A and B) ANOVA showed a significant effect of cytokine treatment on lymphocyte adhesion and transmigration; P < 0.01; *, P < 0.05; **, P < 0.01, compared with untreated by Dunnett test.
Mentions: When PBL were allowed to settle for 5 min, few (∼5%) adhered to unstimulated HUVEC cultured in multiwell plates, but cytokine-stimulated EC supported much higher levels of attachment (Fig. 3A). We were surprised to find that 2 min after washing, a significant proportion of the adherent cells had transmigrated and that after a further 15 min, this proportion remained essentially the same (Fig. 3B). Transmigration was higher for cytokine-treated monolayers, particularly in the presence of IFN. Although some adherent cells did transmigrate through unstimulated HUVEC in minutes, the absolute number observed was small as a result of the low level of adhesion. Examining migration through cytokine-treated EC in more detail, we recorded individual fields and repeatedly assessed transmigration. Although there were minor fluctuations in the proportion of PBL transmigrated, there were no significant upward trends (Fig. 3C). We also noted the velocity of migrated cells, which averaged ∼8 μm/min and tended to be faster in the presence of IFN (Table 1A). Thus, in a static assay with short initial contact times, we detected cytokine-specific induction of lymphocyte adhesion and could observe transendothelial migration within minutes.

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