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Transcellular migration of leukocytes is mediated by the endothelial lateral border recycling compartment.

Mamdouh Z, Mikhailov A, Muller WA - J. Exp. Med. (2009)

Bottom Line: Depolymerization of microtubules has no effect on ICAM-1 enrichment but blocks targeted trafficking of LBRC membrane and transcellular migration by >90%.Similar to their effects on paracellular transmigration, antibodies against PECAM or CD99, but not JAM-A, block transcellular migration.We conclude that similar molecular mechanisms promote both para- and transcellular migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

ABSTRACT
Leukocyte migration across endothelial cell borders (paracellular) and through endothelial cells (transcellular) appear to be distinct processes. During paracellular migration, membrane from a parajunctional reticulum of interconnected vesicles, the endothelial lateral border recycling compartment (LBRC), moves to surround the leukocyte in a kinesin-mediated, microtubule-dependent manner. We show that transcellular migration likewise requires targeted trafficking of LBRC membrane. We show that in addition to platelet/endothelial cell adhesion molecule (PECAM; CD31), CD99 and junctional adhesion molecule A (JAM-A), but apparently not vascular endothelial cell-specific cadherin (cadherin 5, CD144), are components of the LBRC. During transcellular migration, LBRC membrane invests the transmigrating leukocyte. Intracellular adhesion molecule 1 (ICAM-1) on the apical endothelial surface is enriched around adherent leukocytes. Depolymerization of microtubules has no effect on ICAM-1 enrichment but blocks targeted trafficking of LBRC membrane and transcellular migration by >90%. Similar to their effects on paracellular transmigration, antibodies against PECAM or CD99, but not JAM-A, block transcellular migration. We conclude that similar molecular mechanisms promote both para- and transcellular migration.

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Blocking PECAM or CD99 blocks transcellular migration. Transmigration assays conducted in the presence of antibodies against PECAM, CD99, JAM-A, or VE-cadherin and analyzed as described in Materials and methods. The mAb against VE-cadherin was used as a negative control, because it has been shown not to inhibit transmigration (Muller et al., 1993; Schenkel et al., 2002). Leukocytes were scored as nontransmigrated, transmigrated paracellularly (Para), or transmigrated transcellularly (Trans). Under these conditions, ∼80% of control leukocytes transmigrated: ∼50% paracellularly and 30% transcellularly. Antibodies against PECAM and CD99 blocked both para- and transcellular diapedesis significantly, whereas antibody against JAM-A had no effect. Data are means ± SEM from three separate experiments in which >250 leukocytes were counted for each condition. *, P < 10−5 compared with VE-cadherin control or JAM-A.
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fig6: Blocking PECAM or CD99 blocks transcellular migration. Transmigration assays conducted in the presence of antibodies against PECAM, CD99, JAM-A, or VE-cadherin and analyzed as described in Materials and methods. The mAb against VE-cadherin was used as a negative control, because it has been shown not to inhibit transmigration (Muller et al., 1993; Schenkel et al., 2002). Leukocytes were scored as nontransmigrated, transmigrated paracellularly (Para), or transmigrated transcellularly (Trans). Under these conditions, ∼80% of control leukocytes transmigrated: ∼50% paracellularly and 30% transcellularly. Antibodies against PECAM and CD99 blocked both para- and transcellular diapedesis significantly, whereas antibody against JAM-A had no effect. Data are means ± SEM from three separate experiments in which >250 leukocytes were counted for each condition. *, P < 10−5 compared with VE-cadherin control or JAM-A.

Mentions: Paracellular transmigration of human neutrophils and monocytes, which requires targeted recycling of LBRC membrane (Mamdouh et al., 2003; Mamdouh et al., 2008), can be blocked by mAbs against PECAM and CD99 but not JAM-A (Muller et al., 1993; Liu et al., 2000; Schenkel et al., 2002; Schenkel et al., 2004). This raised the question of whether transcellular migration similarly could be blocked by these antibodies. Transmigration assays were performed in the presence of antibodies against PECAM, CD99, JAM-A, or VE-cadherin and examined by confocal microscopy, scoring for leukocytes that had undergone para- or transcellular diapedesis (Fig. 6). To be able to distinguish leukocytes blocked in the act of transmigration from those that were actually going through (especially for CD99, which arrests leukocytes part way through the junction; Schenkel et al., 2002; Lou et al., 2007), we ran the transmigration assay for 30 min to allow ample time for unblocked leukocytes to move across the endothelial monolayer. As a result, the control transmigration rate was ∼80%, with up to 33% of the leukocytes migrating transcellularly (compare with the 10-min assay in Fig. 4 in which only ∼45% of control leukocytes transmigrated.) As previously reported, paracellular diapedesis was effectively blocked (by ∼75% compared with control) by antibody against PECAM or CD99 but not against JAM-A or VE-cadherin (Muller et al., 1993; Liu et al., 2000; Schenkel et al., 2002; Schenkel et al., 2004). Interestingly, transcellular diapedesis was also blocked by antibody against PECAM or CD99 but not against JAM-A or VE-cadherin, and transcellular diapedesis was inhibited to about the same extent as was paracellular diapedesis. In pilot experiments, the block in transcellular migration was confirmed by live-cell imaging using differential interference contrast optics (Schenkel et al., 2004; Lou et al., 2007).


Transcellular migration of leukocytes is mediated by the endothelial lateral border recycling compartment.

Mamdouh Z, Mikhailov A, Muller WA - J. Exp. Med. (2009)

Blocking PECAM or CD99 blocks transcellular migration. Transmigration assays conducted in the presence of antibodies against PECAM, CD99, JAM-A, or VE-cadherin and analyzed as described in Materials and methods. The mAb against VE-cadherin was used as a negative control, because it has been shown not to inhibit transmigration (Muller et al., 1993; Schenkel et al., 2002). Leukocytes were scored as nontransmigrated, transmigrated paracellularly (Para), or transmigrated transcellularly (Trans). Under these conditions, ∼80% of control leukocytes transmigrated: ∼50% paracellularly and 30% transcellularly. Antibodies against PECAM and CD99 blocked both para- and transcellular diapedesis significantly, whereas antibody against JAM-A had no effect. Data are means ± SEM from three separate experiments in which >250 leukocytes were counted for each condition. *, P < 10−5 compared with VE-cadherin control or JAM-A.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2806621&req=5

fig6: Blocking PECAM or CD99 blocks transcellular migration. Transmigration assays conducted in the presence of antibodies against PECAM, CD99, JAM-A, or VE-cadherin and analyzed as described in Materials and methods. The mAb against VE-cadherin was used as a negative control, because it has been shown not to inhibit transmigration (Muller et al., 1993; Schenkel et al., 2002). Leukocytes were scored as nontransmigrated, transmigrated paracellularly (Para), or transmigrated transcellularly (Trans). Under these conditions, ∼80% of control leukocytes transmigrated: ∼50% paracellularly and 30% transcellularly. Antibodies against PECAM and CD99 blocked both para- and transcellular diapedesis significantly, whereas antibody against JAM-A had no effect. Data are means ± SEM from three separate experiments in which >250 leukocytes were counted for each condition. *, P < 10−5 compared with VE-cadherin control or JAM-A.
Mentions: Paracellular transmigration of human neutrophils and monocytes, which requires targeted recycling of LBRC membrane (Mamdouh et al., 2003; Mamdouh et al., 2008), can be blocked by mAbs against PECAM and CD99 but not JAM-A (Muller et al., 1993; Liu et al., 2000; Schenkel et al., 2002; Schenkel et al., 2004). This raised the question of whether transcellular migration similarly could be blocked by these antibodies. Transmigration assays were performed in the presence of antibodies against PECAM, CD99, JAM-A, or VE-cadherin and examined by confocal microscopy, scoring for leukocytes that had undergone para- or transcellular diapedesis (Fig. 6). To be able to distinguish leukocytes blocked in the act of transmigration from those that were actually going through (especially for CD99, which arrests leukocytes part way through the junction; Schenkel et al., 2002; Lou et al., 2007), we ran the transmigration assay for 30 min to allow ample time for unblocked leukocytes to move across the endothelial monolayer. As a result, the control transmigration rate was ∼80%, with up to 33% of the leukocytes migrating transcellularly (compare with the 10-min assay in Fig. 4 in which only ∼45% of control leukocytes transmigrated.) As previously reported, paracellular diapedesis was effectively blocked (by ∼75% compared with control) by antibody against PECAM or CD99 but not against JAM-A or VE-cadherin (Muller et al., 1993; Liu et al., 2000; Schenkel et al., 2002; Schenkel et al., 2004). Interestingly, transcellular diapedesis was also blocked by antibody against PECAM or CD99 but not against JAM-A or VE-cadherin, and transcellular diapedesis was inhibited to about the same extent as was paracellular diapedesis. In pilot experiments, the block in transcellular migration was confirmed by live-cell imaging using differential interference contrast optics (Schenkel et al., 2004; Lou et al., 2007).

Bottom Line: Depolymerization of microtubules has no effect on ICAM-1 enrichment but blocks targeted trafficking of LBRC membrane and transcellular migration by >90%.Similar to their effects on paracellular transmigration, antibodies against PECAM or CD99, but not JAM-A, block transcellular migration.We conclude that similar molecular mechanisms promote both para- and transcellular migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.

ABSTRACT
Leukocyte migration across endothelial cell borders (paracellular) and through endothelial cells (transcellular) appear to be distinct processes. During paracellular migration, membrane from a parajunctional reticulum of interconnected vesicles, the endothelial lateral border recycling compartment (LBRC), moves to surround the leukocyte in a kinesin-mediated, microtubule-dependent manner. We show that transcellular migration likewise requires targeted trafficking of LBRC membrane. We show that in addition to platelet/endothelial cell adhesion molecule (PECAM; CD31), CD99 and junctional adhesion molecule A (JAM-A), but apparently not vascular endothelial cell-specific cadherin (cadherin 5, CD144), are components of the LBRC. During transcellular migration, LBRC membrane invests the transmigrating leukocyte. Intracellular adhesion molecule 1 (ICAM-1) on the apical endothelial surface is enriched around adherent leukocytes. Depolymerization of microtubules has no effect on ICAM-1 enrichment but blocks targeted trafficking of LBRC membrane and transcellular migration by >90%. Similar to their effects on paracellular transmigration, antibodies against PECAM or CD99, but not JAM-A, block transcellular migration. We conclude that similar molecular mechanisms promote both para- and transcellular migration.

Show MeSH
Related in: MedlinePlus