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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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ICAM-1 projections are highly associated with transmigrating cells. Monocytes (A) and neutrophils and lymphocytes (B) were incubated with TNF-α–activated and either MCP-1–, PAF-, or SDF-1–pretreated HUVEC monolayers, respectively, for the indicated number of minutes (m). Cells were fixed and stained for ICAM-1, LFA-1, and VE-cadherin. In each of three to eight separate experiments, a minimum of 100 leukocytes from randomly selected fields for each time point were carefully analyzed in all apical to basal planes and scored for the presence of 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 including TEM stages 1–3) (T) or under (U) the HUVEC monolayer. Each bar represents the percentage of total cells scored at each indicated time point that were found in each of the three categories (i.e., A, T, or U). The black portion of each bar is the fraction of cells scored 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. Values represent mean ± SEM (n = 3–8).
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fig4: ICAM-1 projections are highly associated with transmigrating cells. Monocytes (A) and neutrophils and lymphocytes (B) were incubated with TNF-α–activated and either MCP-1–, PAF-, or SDF-1–pretreated HUVEC monolayers, respectively, for the indicated number of minutes (m). Cells were fixed and stained for ICAM-1, LFA-1, and VE-cadherin. In each of three to eight separate experiments, a minimum of 100 leukocytes from randomly selected fields for each time point were carefully analyzed in all apical to basal planes and scored for the presence of 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 including TEM stages 1–3) (T) or under (U) the HUVEC monolayer. Each bar represents the percentage of total cells scored at each indicated time point that were found in each of the three categories (i.e., A, T, or U). The black portion of each bar is the fraction of cells scored 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. Values represent mean ± SEM (n = 3–8).

Mentions: Over a 60-min time course, the fraction of monocytes that had completed diapedesis steadily increased (Fig. 4 A). At all time points the population of cells that were in any stage of TEM (TEM-1, -2, or -3) was highly associated with ICAM-1 projections (average projection-positive fraction was 96%). Consistent with our previous findings (Carman et al., 2003), a proportion of cells on the apical surface of the endothelium that had not yet initiated diapedesis were also associated with ICAM-1 projections. However, the projection-positive fraction of these cells averaged only 41% (P value vs. transmigrating cells of < 0.0001) over the 60-min time course. Using a single time point, a similar analysis was performed on neutrophils and lymphocytes (Fig. 4 B), which demonstrated a nearly complete association of TEM events with ICAM-1 projections (neutrophils, 94%; lymphocytes, 98%) and a significantly lower percentage of adherent, nontransmigrating leukocytes associated with projections (neutrophils, 22% (P < 0.0001); lymphocytes, 23% (P < 0.0001)). This situation was similar under shear conditions (4 dyne/cm2), with the majority of transmigrating monocytes (94 ± 2%), neutrophils, (87 ± 6%) and lymphocytes (91 ± 2) associated with projections.


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

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

ICAM-1 projections are highly associated with transmigrating cells. Monocytes (A) and neutrophils and lymphocytes (B) were incubated with TNF-α–activated and either MCP-1–, PAF-, or SDF-1–pretreated HUVEC monolayers, respectively, for the indicated number of minutes (m). Cells were fixed and stained for ICAM-1, LFA-1, and VE-cadherin. In each of three to eight separate experiments, a minimum of 100 leukocytes from randomly selected fields for each time point were carefully analyzed in all apical to basal planes and scored for the presence of 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 including TEM stages 1–3) (T) or under (U) the HUVEC monolayer. Each bar represents the percentage of total cells scored at each indicated time point that were found in each of the three categories (i.e., A, T, or U). The black portion of each bar is the fraction of cells scored 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. Values represent mean ± SEM (n = 3–8).
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Related In: Results  -  Collection

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fig4: ICAM-1 projections are highly associated with transmigrating cells. Monocytes (A) and neutrophils and lymphocytes (B) were incubated with TNF-α–activated and either MCP-1–, PAF-, or SDF-1–pretreated HUVEC monolayers, respectively, for the indicated number of minutes (m). Cells were fixed and stained for ICAM-1, LFA-1, and VE-cadherin. In each of three to eight separate experiments, a minimum of 100 leukocytes from randomly selected fields for each time point were carefully analyzed in all apical to basal planes and scored for the presence of 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 including TEM stages 1–3) (T) or under (U) the HUVEC monolayer. Each bar represents the percentage of total cells scored at each indicated time point that were found in each of the three categories (i.e., A, T, or U). The black portion of each bar is the fraction of cells scored 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. Values represent mean ± SEM (n = 3–8).
Mentions: Over a 60-min time course, the fraction of monocytes that had completed diapedesis steadily increased (Fig. 4 A). At all time points the population of cells that were in any stage of TEM (TEM-1, -2, or -3) was highly associated with ICAM-1 projections (average projection-positive fraction was 96%). Consistent with our previous findings (Carman et al., 2003), a proportion of cells on the apical surface of the endothelium that had not yet initiated diapedesis were also associated with ICAM-1 projections. However, the projection-positive fraction of these cells averaged only 41% (P value vs. transmigrating cells of < 0.0001) over the 60-min time course. Using a single time point, a similar analysis was performed on neutrophils and lymphocytes (Fig. 4 B), which demonstrated a nearly complete association of TEM events with ICAM-1 projections (neutrophils, 94%; lymphocytes, 98%) and a significantly lower percentage of adherent, nontransmigrating leukocytes associated with projections (neutrophils, 22% (P < 0.0001); lymphocytes, 23% (P < 0.0001)). This situation was similar under shear conditions (4 dyne/cm2), with the majority of transmigrating monocytes (94 ± 2%), neutrophils, (87 ± 6%) and lymphocytes (91 ± 2) associated with projections.

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