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A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them.

Carman CV, Springer TA - J. Cell Biol. (2004)

Bottom Line: We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel "cuplike" transmigratory structure.Disruption of projections was highly correlated with inhibition of transmigration.These findings suggest a novel mechanism, the "transmigratory cup", by which the endothelium provides directional guidance to leukocytes for extravasation.

View Article: PubMed Central - PubMed

Affiliation: The CBR Institute for Biomedical Research, Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The basic route and mechanisms for leukocyte migration across the endothelium remain poorly defined. We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel "cuplike" transmigratory structure. This endothelial structure was comprised of highly intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-enriched vertical microvilli-like projections that surrounded transmigrating leukocytes and drove redistribution of their integrins into linear tracks oriented parallel to the direction of diapedesis. Disruption of projections was highly correlated with inhibition of transmigration. These findings suggest a novel mechanism, the "transmigratory cup", by which the endothelium provides directional guidance to leukocytes for extravasation.

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Asymmetric projections are associated with laterally migrating cells. Monocytes (A) and lymphocytes (B and C) were incubated with TNF-α–activated and either MCP-1– or SDF-1–pretreated HUVEC monolayers, respectively. (A) After 10 min of incubation cells were fixed and stained for ICAM-1 (green) and LFA-1 (red). Four representative top view images depict apically adherent monocytes with asymmetrically distributed ICAM-1 projections. (B) Live-cell imaging of a representative laterally migrating SNARF-labeled lymphocyte (red) on the apical surface of the endothelium prelabeled with an anti–ICAM-1–Alexa 488 Fab fragment (green). Image depicts top (0°; top) and side (90°; bottom) views of a single ICAM-1–bearing cellular projection that is largely suspended above the apical surface of the endothelium and is attached to a lymphocyte migrating laterally to the right. (C) Live-cell time lapse imaging of another cell as in B. ICAM-1 projections emanating from the apical surface of the endothelium (arrowheads) attached to the trailing edge of a lymphocyte become elongated as the lymphocyte advances. Panels depict top view projections of confocal sections at 0, 2, and 4 min as indicated. Bars, 5 μm.
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fig5: Asymmetric projections are associated with laterally migrating cells. Monocytes (A) and lymphocytes (B and C) were incubated with TNF-α–activated and either MCP-1– or SDF-1–pretreated HUVEC monolayers, respectively. (A) After 10 min of incubation cells were fixed and stained for ICAM-1 (green) and LFA-1 (red). Four representative top view images depict apically adherent monocytes with asymmetrically distributed ICAM-1 projections. (B) Live-cell imaging of a representative laterally migrating SNARF-labeled lymphocyte (red) on the apical surface of the endothelium prelabeled with an anti–ICAM-1–Alexa 488 Fab fragment (green). Image depicts top (0°; top) and side (90°; bottom) views of a single ICAM-1–bearing cellular projection that is largely suspended above the apical surface of the endothelium and is attached to a lymphocyte migrating laterally to the right. (C) Live-cell time lapse imaging of another cell as in B. ICAM-1 projections emanating from the apical surface of the endothelium (arrowheads) attached to the trailing edge of a lymphocyte become elongated as the lymphocyte advances. Panels depict top view projections of confocal sections at 0, 2, and 4 min as indicated. Bars, 5 μm.

Mentions: Of the ICAM-1 projection-positive population of transmigrating leukocytes, over all time points, 97% were associated with cuplike projections that were largely symmetrical, encircling at least 240° of the circumference of the leukocytes (See Fig. 6, A–C, bottom; Fig. S4, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200404129/DC1). In contrast, of the projection-positive, apically adherent, nontransmigrating leukocytes, 30–50% were associated with asymmetrically distributed, “tetherlike” projections that contacted only one side of the leukocytes and were extended several, and sometimes up to 30 microns in length (Fig. 5). In these cases the leukocytes frequently exhibited polarized shapes suggestive of lateral migration (Worthylake and Burridge, 2001; Fig. 5). The attachment point on the leukocyte was typically one to several microns above the apical surface of the endothelium, and views parallel to the substrate (Fig. 5 B, bottom) demonstrated that the projections spanned the distance between their endothelial origin and the leukocyte suspended above the endothelium. This architecture is roughly analogous to that described for selectin-mediated transient tethers formed under shear flow, with the distinction that tethers extended up from the endothelium to the reach leukocytes rather than down from the leukocyte to reach the substrate (Chen and Springer, 1999). These findings strongly suggest that tetherlike projections were asymmetric as a result of lateral leukocyte migration away from their origin on the endothelium. To confirm this, live-cell time-lapse confocal microscopy was performed. This demonstrated that the distal ends of the projections were, indeed, attached to the trailing edge of the leukocytes and extended as a consequence of leukocyte lateral migration, whereas the endothelial origin of the projections remained stationary (Fig. 5 C). Conversely, time-lapse showed that symmetric projections formed around leukocytes that were not migrating laterally (unpublished data).


A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them.

Carman CV, Springer TA - J. Cell Biol. (2004)

Asymmetric projections are associated with laterally migrating cells. Monocytes (A) and lymphocytes (B and C) were incubated with TNF-α–activated and either MCP-1– or SDF-1–pretreated HUVEC monolayers, respectively. (A) After 10 min of incubation cells were fixed and stained for ICAM-1 (green) and LFA-1 (red). Four representative top view images depict apically adherent monocytes with asymmetrically distributed ICAM-1 projections. (B) Live-cell imaging of a representative laterally migrating SNARF-labeled lymphocyte (red) on the apical surface of the endothelium prelabeled with an anti–ICAM-1–Alexa 488 Fab fragment (green). Image depicts top (0°; top) and side (90°; bottom) views of a single ICAM-1–bearing cellular projection that is largely suspended above the apical surface of the endothelium and is attached to a lymphocyte migrating laterally to the right. (C) Live-cell time lapse imaging of another cell as in B. ICAM-1 projections emanating from the apical surface of the endothelium (arrowheads) attached to the trailing edge of a lymphocyte become elongated as the lymphocyte advances. Panels depict top view projections of confocal sections at 0, 2, and 4 min as indicated. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172560&req=5

fig5: Asymmetric projections are associated with laterally migrating cells. Monocytes (A) and lymphocytes (B and C) were incubated with TNF-α–activated and either MCP-1– or SDF-1–pretreated HUVEC monolayers, respectively. (A) After 10 min of incubation cells were fixed and stained for ICAM-1 (green) and LFA-1 (red). Four representative top view images depict apically adherent monocytes with asymmetrically distributed ICAM-1 projections. (B) Live-cell imaging of a representative laterally migrating SNARF-labeled lymphocyte (red) on the apical surface of the endothelium prelabeled with an anti–ICAM-1–Alexa 488 Fab fragment (green). Image depicts top (0°; top) and side (90°; bottom) views of a single ICAM-1–bearing cellular projection that is largely suspended above the apical surface of the endothelium and is attached to a lymphocyte migrating laterally to the right. (C) Live-cell time lapse imaging of another cell as in B. ICAM-1 projections emanating from the apical surface of the endothelium (arrowheads) attached to the trailing edge of a lymphocyte become elongated as the lymphocyte advances. Panels depict top view projections of confocal sections at 0, 2, and 4 min as indicated. Bars, 5 μm.
Mentions: Of the ICAM-1 projection-positive population of transmigrating leukocytes, over all time points, 97% were associated with cuplike projections that were largely symmetrical, encircling at least 240° of the circumference of the leukocytes (See Fig. 6, A–C, bottom; Fig. S4, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200404129/DC1). In contrast, of the projection-positive, apically adherent, nontransmigrating leukocytes, 30–50% were associated with asymmetrically distributed, “tetherlike” projections that contacted only one side of the leukocytes and were extended several, and sometimes up to 30 microns in length (Fig. 5). In these cases the leukocytes frequently exhibited polarized shapes suggestive of lateral migration (Worthylake and Burridge, 2001; Fig. 5). The attachment point on the leukocyte was typically one to several microns above the apical surface of the endothelium, and views parallel to the substrate (Fig. 5 B, bottom) demonstrated that the projections spanned the distance between their endothelial origin and the leukocyte suspended above the endothelium. This architecture is roughly analogous to that described for selectin-mediated transient tethers formed under shear flow, with the distinction that tethers extended up from the endothelium to the reach leukocytes rather than down from the leukocyte to reach the substrate (Chen and Springer, 1999). These findings strongly suggest that tetherlike projections were asymmetric as a result of lateral leukocyte migration away from their origin on the endothelium. To confirm this, live-cell time-lapse confocal microscopy was performed. This demonstrated that the distal ends of the projections were, indeed, attached to the trailing edge of the leukocytes and extended as a consequence of leukocyte lateral migration, whereas the endothelial origin of the projections remained stationary (Fig. 5 C). Conversely, time-lapse showed that symmetric projections formed around leukocytes that were not migrating laterally (unpublished data).

Bottom Line: We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel "cuplike" transmigratory structure.Disruption of projections was highly correlated with inhibition of transmigration.These findings suggest a novel mechanism, the "transmigratory cup", by which the endothelium provides directional guidance to leukocytes for extravasation.

View Article: PubMed Central - PubMed

Affiliation: The CBR Institute for Biomedical Research, Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
The basic route and mechanisms for leukocyte migration across the endothelium remain poorly defined. We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel "cuplike" transmigratory structure. This endothelial structure was comprised of highly intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-enriched vertical microvilli-like projections that surrounded transmigrating leukocytes and drove redistribution of their integrins into linear tracks oriented parallel to the direction of diapedesis. Disruption of projections was highly correlated with inhibition of transmigration. These findings suggest a novel mechanism, the "transmigratory cup", by which the endothelium provides directional guidance to leukocytes for extravasation.

Show MeSH
Related in: MedlinePlus