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Direct evidence for activated CD8+ T cell transmigration across portal vein endothelial cells in liver graft rejection

View Article: PubMed Central - PubMed

ABSTRACT

Background: Lymphocyte recruitment into the portal tract is crucial not only for homeostatic immune surveillance but also for many liver diseases. However, the exact route of entry for lymphocytes into portal tract is still obscure. We investigated this question using a rat hepatic allograft rejection model.

Methods: A migration route was analyzed by immunohistological methods including a recently developed scanning electron microscopy method. Transmigration-associated molecules such as selectins, integrins, and chemokines and their receptors expressed by hepatic vessels and recruited T-cells were analyzed by immunohistochemistry and flow cytometry.

Results: The immunoelectron microscopic analysis clearly showed CD8β+ cells passing through the portal vein (PV) endothelia. Furthermore, the migrating pathway seemed to pass through the endothelial cell body. Local vascular cell adhesion molecule-1 (VCAM-1) expression was induced in PV endothelial cells from day 2 after liver transplantation. Although intercellular adhesion molecule-1 (ICAM-1) expression was also upregulated, it was restricted to sinusoidal endothelia. Recipient T-cells in the graft perfusate were CD25+CD44+ICAM-1+CXCR3+CCR5– and upregulated α4β1 or αLβ2 integrins. Immunohistochemistry showed the expression of CXCL10 in donor MHCIIhigh cells in the portal tract as well as endothelial walls of PV.

Conclusions: We show for the first time direct evidence of T-cell transmigration across PV endothelial cells during hepatic allograft rejection. Interactions between VCAM-1 on endothelia and α4β1 integrin on recipient effector T-cells putatively play critical roles in adhesion and transmigration through endothelia. A chemokine axis of CXCL10 and CXCR3 also may be involved.

Electronic supplementary material: The online version of this article (doi:10.1007/s00535-016-1169-1) contains supplementary material, which is available to authorized users.

No MeSH data available.


SEM images of the portal tract of the allograft. Representative SEM images of the PV (a–i) and hepatic vein (j–l) after LTx. Note the appearance of adherent cells from day 2 (b, h) in Fig. 1. Note poorly polarized cells, with a less protrusional shape of adherent cells at the PV (e, f) compared to those of hepatic vein (k, l). Immuno-SEM analysis for CD8β (m–r). Note CD8β+ cells undergoing transmigration at the PV (m and n, black arrowhead). A backscatter electron (BSE) image of 1–3 (o–r). Note considerable amount of CD8β signal was evenly and densely distributed in the cell body (q) as well as the lamellipodia-like structure (r, white arrowheads) of 1, but not 2 and 3 (o and p). Bd bile duct, PV portal vein, HA hepatic artery, HV hepatic vein. Scale bars: a, c, d, j, and m 50 μm; b, g 30 μm; h, i 10 μm; n–p 2.5 μm; q 2 μm; r 0.5 μm
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Fig2: SEM images of the portal tract of the allograft. Representative SEM images of the PV (a–i) and hepatic vein (j–l) after LTx. Note the appearance of adherent cells from day 2 (b, h) in Fig. 1. Note poorly polarized cells, with a less protrusional shape of adherent cells at the PV (e, f) compared to those of hepatic vein (k, l). Immuno-SEM analysis for CD8β (m–r). Note CD8β+ cells undergoing transmigration at the PV (m and n, black arrowhead). A backscatter electron (BSE) image of 1–3 (o–r). Note considerable amount of CD8β signal was evenly and densely distributed in the cell body (q) as well as the lamellipodia-like structure (r, white arrowheads) of 1, but not 2 and 3 (o and p). Bd bile duct, PV portal vein, HA hepatic artery, HV hepatic vein. Scale bars: a, c, d, j, and m 50 μm; b, g 30 μm; h, i 10 μm; n–p 2.5 μm; q 2 μm; r 0.5 μm

Mentions: Immunohistochemical analysis showed that some cells attached on the wall of the PV (Fig. 1h, i). SEM imaging of the allograft showed that the number of leukocytes contacting the vessel wall gradually increased from day 2 at the portal tract (Fig. 2a–i). Of interest, their shapes were obviously different from those in the hepatic vein, with a spherical, non-polarized morphology (Fig. 2d–f) compared to a non-spherical morphology with spreading microvilli in the latter (Fig. 2j–l). Many bulges were also formed on the vessel wall compared to the control group, implying the presence of migrating lymphocytes underneath the endothelial sheet (red asterisk, in Fig. 2i). Furthermore, by immuno-SEM analysis using the anti-CD8β mAb followed by nano-gold–conjugated secondary antibody, we could detect CD8β+ particles on a cell that was just passing through the PV endothelial cell (Fig. 2m, n, q, and r). We could not investigate their transmigration of CD4+ T-cells because of a lack of anti-rat CD4 mAb compatible with 4 % paraformaldehyde fixation, an essential procedure for immuno-SEM analysis.Fig. 2


Direct evidence for activated CD8+ T cell transmigration across portal vein endothelial cells in liver graft rejection
SEM images of the portal tract of the allograft. Representative SEM images of the PV (a–i) and hepatic vein (j–l) after LTx. Note the appearance of adherent cells from day 2 (b, h) in Fig. 1. Note poorly polarized cells, with a less protrusional shape of adherent cells at the PV (e, f) compared to those of hepatic vein (k, l). Immuno-SEM analysis for CD8β (m–r). Note CD8β+ cells undergoing transmigration at the PV (m and n, black arrowhead). A backscatter electron (BSE) image of 1–3 (o–r). Note considerable amount of CD8β signal was evenly and densely distributed in the cell body (q) as well as the lamellipodia-like structure (r, white arrowheads) of 1, but not 2 and 3 (o and p). Bd bile duct, PV portal vein, HA hepatic artery, HV hepatic vein. Scale bars: a, c, d, j, and m 50 μm; b, g 30 μm; h, i 10 μm; n–p 2.5 μm; q 2 μm; r 0.5 μm
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Fig2: SEM images of the portal tract of the allograft. Representative SEM images of the PV (a–i) and hepatic vein (j–l) after LTx. Note the appearance of adherent cells from day 2 (b, h) in Fig. 1. Note poorly polarized cells, with a less protrusional shape of adherent cells at the PV (e, f) compared to those of hepatic vein (k, l). Immuno-SEM analysis for CD8β (m–r). Note CD8β+ cells undergoing transmigration at the PV (m and n, black arrowhead). A backscatter electron (BSE) image of 1–3 (o–r). Note considerable amount of CD8β signal was evenly and densely distributed in the cell body (q) as well as the lamellipodia-like structure (r, white arrowheads) of 1, but not 2 and 3 (o and p). Bd bile duct, PV portal vein, HA hepatic artery, HV hepatic vein. Scale bars: a, c, d, j, and m 50 μm; b, g 30 μm; h, i 10 μm; n–p 2.5 μm; q 2 μm; r 0.5 μm
Mentions: Immunohistochemical analysis showed that some cells attached on the wall of the PV (Fig. 1h, i). SEM imaging of the allograft showed that the number of leukocytes contacting the vessel wall gradually increased from day 2 at the portal tract (Fig. 2a–i). Of interest, their shapes were obviously different from those in the hepatic vein, with a spherical, non-polarized morphology (Fig. 2d–f) compared to a non-spherical morphology with spreading microvilli in the latter (Fig. 2j–l). Many bulges were also formed on the vessel wall compared to the control group, implying the presence of migrating lymphocytes underneath the endothelial sheet (red asterisk, in Fig. 2i). Furthermore, by immuno-SEM analysis using the anti-CD8β mAb followed by nano-gold–conjugated secondary antibody, we could detect CD8β+ particles on a cell that was just passing through the PV endothelial cell (Fig. 2m, n, q, and r). We could not investigate their transmigration of CD4+ T-cells because of a lack of anti-rat CD4 mAb compatible with 4 % paraformaldehyde fixation, an essential procedure for immuno-SEM analysis.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Lymphocyte recruitment into the portal tract is crucial not only for homeostatic immune surveillance but also for many liver diseases. However, the exact route of entry for lymphocytes into portal tract is still obscure. We investigated this question using a rat hepatic allograft rejection model.

Methods: A migration route was analyzed by immunohistological methods including a recently developed scanning electron microscopy method. Transmigration-associated molecules such as selectins, integrins, and chemokines and their receptors expressed by hepatic vessels and recruited T-cells were analyzed by immunohistochemistry and flow cytometry.

Results: The immunoelectron microscopic analysis clearly showed CD8β+ cells passing through the portal vein (PV) endothelia. Furthermore, the migrating pathway seemed to pass through the endothelial cell body. Local vascular cell adhesion molecule-1 (VCAM-1) expression was induced in PV endothelial cells from day 2 after liver transplantation. Although intercellular adhesion molecule-1 (ICAM-1) expression was also upregulated, it was restricted to sinusoidal endothelia. Recipient T-cells in the graft perfusate were CD25+CD44+ICAM-1+CXCR3+CCR5– and upregulated α4β1 or αLβ2 integrins. Immunohistochemistry showed the expression of CXCL10 in donor MHCIIhigh cells in the portal tract as well as endothelial walls of PV.

Conclusions: We show for the first time direct evidence of T-cell transmigration across PV endothelial cells during hepatic allograft rejection. Interactions between VCAM-1 on endothelia and α4β1 integrin on recipient effector T-cells putatively play critical roles in adhesion and transmigration through endothelia. A chemokine axis of CXCL10 and CXCR3 also may be involved.

Electronic supplementary material: The online version of this article (doi:10.1007/s00535-016-1169-1) contains supplementary material, which is available to authorized users.

No MeSH data available.