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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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Inhibition of ICAM-1 projections by BAPTA-AM, colchicine, or toxin-B is highly correlated with reduced TEM. (A) TNF-α–activated, MCP-1–pretreated HUVECs were pretreated with vehicle (DMSO, 60 min), 10 μm colchicine (20 min), or 20 μm BAPTA-AM (60 min) washed and incubated with monocytes for 10 min at 37°C. Cells were fixed, stained for ICAM-1 (IC1/11-488), and LFA-1 (CBR-LFA1/7-Cy3) and then analyzed by confocal microscopy. In each of three to five separate experiments at least 100 monocytes in randomly selected fields were carefully analyzed in all apical to basal planes and scored for the presence of significant ICAM-1–enriched projections of 1 μm in length or greater and as being either apically adherent (A), in the process of TEM (including both para- and transcellular events and TEM 1–3) (T) or under (U) the HUVEC monolayer. Each bar represents the percentage of total cells scored for each experimental condition (DMSO, colchicine, or BAPTA-AM) in each of the three categories. The black portion of each bar is the fraction of cells positive for associated ICAM-1 projections. The gray portion of each bar is the fraction of the cells that were negative for the presence of ICAM-1 projections. (B) TNF-α–activated, SDF-1–pretreated HUVECs were pretreated with vehicle (PBS, 60 min, control), 100 ng/ml toxin-B (60 min) or 50 μg/ml C3 transferase (16 h) washed and then incubated with human lymphocytes for 10 min at 37°C. Samples were fixed, stained, and analyzed as in A. Values represent mean ± SEM (n = 3–5).
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fig8: Inhibition of ICAM-1 projections by BAPTA-AM, colchicine, or toxin-B is highly correlated with reduced TEM. (A) TNF-α–activated, MCP-1–pretreated HUVECs were pretreated with vehicle (DMSO, 60 min), 10 μm colchicine (20 min), or 20 μm BAPTA-AM (60 min) washed and incubated with monocytes for 10 min at 37°C. Cells were fixed, stained for ICAM-1 (IC1/11-488), and LFA-1 (CBR-LFA1/7-Cy3) and then analyzed by confocal microscopy. In each of three to five separate experiments at least 100 monocytes in randomly selected fields were carefully analyzed in all apical to basal planes and scored for the presence of significant ICAM-1–enriched projections of 1 μm in length or greater and as being either apically adherent (A), in the process of TEM (including both para- and transcellular events and TEM 1–3) (T) or under (U) the HUVEC monolayer. Each bar represents the percentage of total cells scored for each experimental condition (DMSO, colchicine, or BAPTA-AM) in each of the three categories. The black portion of each bar is the fraction of cells positive for associated ICAM-1 projections. The gray portion of each bar is the fraction of the cells that were negative for the presence of ICAM-1 projections. (B) TNF-α–activated, SDF-1–pretreated HUVECs were pretreated with vehicle (PBS, 60 min, control), 100 ng/ml toxin-B (60 min) or 50 μg/ml C3 transferase (16 h) washed and then incubated with human lymphocytes for 10 min at 37°C. Samples were fixed, stained, and analyzed as in A. Values represent mean ± SEM (n = 3–5).

Mentions: To assess the functional role of the ICAM-1–enriched projections in TEM, we scored the relative proportions of monocytes that were adherent, transmigrating or under the HUVEC monolayer after treatment with reagents (colchicine or 1,2Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); BAPTA-AM) shown previously to inhibit projections (Carman et al., 2003). Compared with controls, colchicine and BAPTA-AM significantly reduced both para- and transcellular TEM (∼3-4-fold; Fig. 8 A), which is consistent with previous studies (Huang et al., 1993; Etienne-Manneville et al., 2000; J. Greenwood and P. Adamson, personal communication). Whereas 50% of adherent cells had ICAM-1 projections on the control monolayers, only 16% and 18% of the adherent cells on colchicine and BAPTA-AM treated monolayers, respectively, were associated with projections (Fig. 8 A). However, among the leukocytes that were in the process of TEM, the majority (96%, 91%, and 90% for control, colchicine, and BAPTA-AM–treated samples, respectively) were associated with ICAM-1 projections, regardless of HUVEC pretreatment (Fig. 8 A).


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

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

Inhibition of ICAM-1 projections by BAPTA-AM, colchicine, or toxin-B is highly correlated with reduced TEM. (A) TNF-α–activated, MCP-1–pretreated HUVECs were pretreated with vehicle (DMSO, 60 min), 10 μm colchicine (20 min), or 20 μm BAPTA-AM (60 min) washed and incubated with monocytes for 10 min at 37°C. Cells were fixed, stained for ICAM-1 (IC1/11-488), and LFA-1 (CBR-LFA1/7-Cy3) and then analyzed by confocal microscopy. In each of three to five separate experiments at least 100 monocytes in randomly selected fields were carefully analyzed in all apical to basal planes and scored for the presence of significant ICAM-1–enriched projections of 1 μm in length or greater and as being either apically adherent (A), in the process of TEM (including both para- and transcellular events and TEM 1–3) (T) or under (U) the HUVEC monolayer. Each bar represents the percentage of total cells scored for each experimental condition (DMSO, colchicine, or BAPTA-AM) in each of the three categories. The black portion of each bar is the fraction of cells positive for associated ICAM-1 projections. The gray portion of each bar is the fraction of the cells that were negative for the presence of ICAM-1 projections. (B) TNF-α–activated, SDF-1–pretreated HUVECs were pretreated with vehicle (PBS, 60 min, control), 100 ng/ml toxin-B (60 min) or 50 μg/ml C3 transferase (16 h) washed and then incubated with human lymphocytes for 10 min at 37°C. Samples were fixed, stained, and analyzed as in A. Values represent mean ± SEM (n = 3–5).
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fig8: Inhibition of ICAM-1 projections by BAPTA-AM, colchicine, or toxin-B is highly correlated with reduced TEM. (A) TNF-α–activated, MCP-1–pretreated HUVECs were pretreated with vehicle (DMSO, 60 min), 10 μm colchicine (20 min), or 20 μm BAPTA-AM (60 min) washed and incubated with monocytes for 10 min at 37°C. Cells were fixed, stained for ICAM-1 (IC1/11-488), and LFA-1 (CBR-LFA1/7-Cy3) and then analyzed by confocal microscopy. In each of three to five separate experiments at least 100 monocytes in randomly selected fields were carefully analyzed in all apical to basal planes and scored for the presence of significant ICAM-1–enriched projections of 1 μm in length or greater and as being either apically adherent (A), in the process of TEM (including both para- and transcellular events and TEM 1–3) (T) or under (U) the HUVEC monolayer. Each bar represents the percentage of total cells scored for each experimental condition (DMSO, colchicine, or BAPTA-AM) in each of the three categories. The black portion of each bar is the fraction of cells positive for associated ICAM-1 projections. The gray portion of each bar is the fraction of the cells that were negative for the presence of ICAM-1 projections. (B) TNF-α–activated, SDF-1–pretreated HUVECs were pretreated with vehicle (PBS, 60 min, control), 100 ng/ml toxin-B (60 min) or 50 μg/ml C3 transferase (16 h) washed and then incubated with human lymphocytes for 10 min at 37°C. Samples were fixed, stained, and analyzed as in A. Values represent mean ± SEM (n = 3–5).
Mentions: To assess the functional role of the ICAM-1–enriched projections in TEM, we scored the relative proportions of monocytes that were adherent, transmigrating or under the HUVEC monolayer after treatment with reagents (colchicine or 1,2Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester); BAPTA-AM) shown previously to inhibit projections (Carman et al., 2003). Compared with controls, colchicine and BAPTA-AM significantly reduced both para- and transcellular TEM (∼3-4-fold; Fig. 8 A), which is consistent with previous studies (Huang et al., 1993; Etienne-Manneville et al., 2000; J. Greenwood and P. Adamson, personal communication). Whereas 50% of adherent cells had ICAM-1 projections on the control monolayers, only 16% and 18% of the adherent cells on colchicine and BAPTA-AM treated monolayers, respectively, were associated with projections (Fig. 8 A). However, among the leukocytes that were in the process of TEM, the majority (96%, 91%, and 90% for control, colchicine, and BAPTA-AM–treated samples, respectively) were associated with ICAM-1 projections, regardless of HUVEC pretreatment (Fig. 8 A).

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