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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.


Flow cytometry analysis of recruited T-cells in the graft vasculature. a Purity of liver perfusate analyzed. b Expression of migration-associated molecules in CD4 and CD8 T-cells in the liver perfusate at day 3 after LTx (red line) and in control (filled gray). c Upregulation of αLβ2 integrin and α4β1 integrin on activated T-cells after LTx. d Induction of β7 integrin in β1 integrinhigh T-cells. Representative figures of more than three experiments
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Fig5: Flow cytometry analysis of recruited T-cells in the graft vasculature. a Purity of liver perfusate analyzed. b Expression of migration-associated molecules in CD4 and CD8 T-cells in the liver perfusate at day 3 after LTx (red line) and in control (filled gray). c Upregulation of αLβ2 integrin and α4β1 integrin on activated T-cells after LTx. d Induction of β7 integrin in β1 integrinhigh T-cells. Representative figures of more than three experiments

Mentions: To confirm the expression of cell migration-associated molecules in recipient migrating cells, recipient cells inside the graft vasculature were isolated and analyzed by multicolor FCM (Fig. 5). Recipient MHCI+ cells were about ~95 % of the population (Fig. 5a). Histological analysis of the perfused liver indicated that the tissue structure was preserved and the infiltrated cells were not washed away from the interstitial area when compared with unperfused tissue. This result suggested that cells in perfusate were obtained mostly from the blood within the graft and that contamination from cells that had already extravasated was minimal (Suppl. Fig. 1a). Recipient cells in the perfusate consisted of T-cells (~30 %), neutrophils (~30 %), macrophages (~20 %), B-cells (~5 %), and natural killer cells (~5 %) (Suppl. Fig. 1b). Recipient T-cells showed a typical activated-cell phenotype with CD25, CD44H and ICAM-1 upregulated and CD62L downregulated (Fig. 5b). In addition, expression of α and β integrins was increased in both CD4 and CD8 T-cells. Of note, almost 60 % of T-cells were αLβ2+ and 25 % were α4β1+ (Fig. 5c). Interestingly, α4β1 expression was dominant in the CD4 subset (CD8 30 %, CD4 70 %, Fig. 5c). In addition, some β1 integrin+ cells significantly co-expressed β7 integrin (Fig. 5d). On the other hand, P-selectin glycoprotein ligand (PSGL)-1, CD15s (Fig. 5b), and α5 integrin (not shown) were not induced in T-cells after LTx. Diapedesis-associated molecules CD38, but not PECAM-1 (CD31) was slightly up-regulated in CD8 T-cells. CD4 T-cells did not express both molecules.Fig. 5


Direct evidence for activated CD8+ T cell transmigration across portal vein endothelial cells in liver graft rejection
Flow cytometry analysis of recruited T-cells in the graft vasculature. a Purity of liver perfusate analyzed. b Expression of migration-associated molecules in CD4 and CD8 T-cells in the liver perfusate at day 3 after LTx (red line) and in control (filled gray). c Upregulation of αLβ2 integrin and α4β1 integrin on activated T-cells after LTx. d Induction of β7 integrin in β1 integrinhigh T-cells. Representative figures of more than three experiments
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Fig5: Flow cytometry analysis of recruited T-cells in the graft vasculature. a Purity of liver perfusate analyzed. b Expression of migration-associated molecules in CD4 and CD8 T-cells in the liver perfusate at day 3 after LTx (red line) and in control (filled gray). c Upregulation of αLβ2 integrin and α4β1 integrin on activated T-cells after LTx. d Induction of β7 integrin in β1 integrinhigh T-cells. Representative figures of more than three experiments
Mentions: To confirm the expression of cell migration-associated molecules in recipient migrating cells, recipient cells inside the graft vasculature were isolated and analyzed by multicolor FCM (Fig. 5). Recipient MHCI+ cells were about ~95 % of the population (Fig. 5a). Histological analysis of the perfused liver indicated that the tissue structure was preserved and the infiltrated cells were not washed away from the interstitial area when compared with unperfused tissue. This result suggested that cells in perfusate were obtained mostly from the blood within the graft and that contamination from cells that had already extravasated was minimal (Suppl. Fig. 1a). Recipient cells in the perfusate consisted of T-cells (~30 %), neutrophils (~30 %), macrophages (~20 %), B-cells (~5 %), and natural killer cells (~5 %) (Suppl. Fig. 1b). Recipient T-cells showed a typical activated-cell phenotype with CD25, CD44H and ICAM-1 upregulated and CD62L downregulated (Fig. 5b). In addition, expression of α and β integrins was increased in both CD4 and CD8 T-cells. Of note, almost 60 % of T-cells were αLβ2+ and 25 % were α4β1+ (Fig. 5c). Interestingly, α4β1 expression was dominant in the CD4 subset (CD8 30 %, CD4 70 %, Fig. 5c). In addition, some β1 integrin+ cells significantly co-expressed β7 integrin (Fig. 5d). On the other hand, P-selectin glycoprotein ligand (PSGL)-1, CD15s (Fig. 5b), and α5 integrin (not shown) were not induced in T-cells after LTx. Diapedesis-associated molecules CD38, but not PECAM-1 (CD31) was slightly up-regulated in CD8 T-cells. CD4 T-cells did not express both molecules.Fig. 5

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.