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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.


Immunohistochemical analysis of cell migration-associated molecule expression in liver allograft endothelia. VCAM-1 and ICAM-1 expression was upregulated after LTx (a–e). Note reciprocal expression pattern for VCAM-1 and ICAM-1 at portal vein and sinusoidal endothelia, respectively (b versus e). VCAM-1 expression was slightly induced in the syngeneic graft (f). The expressions of P-selectin (g and l), PECAM-1 (h, m), VAP-1 (i, n) and tissue fibronectin (k, p, and q, arrowhead) was upregulated while sialyl-6-sulfo-Lewis X (j, o) declined at the PV endothelia after LTx. Scale bars: a, c, and d 200 μm; b, e, and f 100 μm; inset of b, c, e, and f 20 μm; g–p 100 μm; q 20 μm
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Fig4: Immunohistochemical analysis of cell migration-associated molecule expression in liver allograft endothelia. VCAM-1 and ICAM-1 expression was upregulated after LTx (a–e). Note reciprocal expression pattern for VCAM-1 and ICAM-1 at portal vein and sinusoidal endothelia, respectively (b versus e). VCAM-1 expression was slightly induced in the syngeneic graft (f). The expressions of P-selectin (g and l), PECAM-1 (h, m), VAP-1 (i, n) and tissue fibronectin (k, p, and q, arrowhead) was upregulated while sialyl-6-sulfo-Lewis X (j, o) declined at the PV endothelia after LTx. Scale bars: a, c, and d 200 μm; b, e, and f 100 μm; inset of b, c, e, and f 20 μm; g–p 100 μm; q 20 μm

Mentions: Next, we analyzed the expression of cell migration-associated molecules on the graft vascular endothelium by immunohistochemistry. Several cell migration-associated molecules were expressed constitutively and selectively on the PV endothelia in normal donor livers as shown in Table 1. From day 2 after LTx, vascular cell adhesion molecule-1 (VCAM-1) was preferentially induced on the portal but not sinusoidal endothelia and persisted thereafter (Fig. 4a–c). Occasionally, endothelia of the central vein were also partly positive for this molecule. Intercellular cell adhesion molecule-1 (ICAM-1) expression was also upregulated after LTx but was restricted to sinusoidal and hepatic vein endothelia (Fig. 4d, e). Weak expression of VCAM-1 in the PV (Fig. 4f) and of ICAM-1 on sinusoidal endothelia (not shown) was occasionally seen in the syngeneic graft on day 2. P-selectin, platelet/endothelial cell adhesion molecule (PECAM)-1, and vascular adhesion protein (VAP)-1 were constitutively expressed and slightly upregulated in the PV endothelia after LTx (Fig. 4g–i, l–n). In contrast, the expression of sialyl-6-sulfo Lewis X (CD15s), a ligand of selectins, was downregulated in the PV endothelia from day 2 after LTx (Fig. 4j–o). Tissue fibronectin (tFN), one of the ligands of α4β1 integrin, which is upregulated after LTx [12], was temporarily expressed in the PV endothelia from day 3 after LTx (Fig. 4k, p and q). E-selectin expression was unchanged, and neither mucosal vascular addressing cell adhesion molecule-1 (MAdCAM-1) nor ICAM-2 was detected on the surface of the graft vasculature (not shown). The spatio-temporal expression profile of these molecules is summarized in Table 1.Table 1


Direct evidence for activated CD8+ T cell transmigration across portal vein endothelial cells in liver graft rejection
Immunohistochemical analysis of cell migration-associated molecule expression in liver allograft endothelia. VCAM-1 and ICAM-1 expression was upregulated after LTx (a–e). Note reciprocal expression pattern for VCAM-1 and ICAM-1 at portal vein and sinusoidal endothelia, respectively (b versus e). VCAM-1 expression was slightly induced in the syngeneic graft (f). The expressions of P-selectin (g and l), PECAM-1 (h, m), VAP-1 (i, n) and tissue fibronectin (k, p, and q, arrowhead) was upregulated while sialyl-6-sulfo-Lewis X (j, o) declined at the PV endothelia after LTx. Scale bars: a, c, and d 200 μm; b, e, and f 100 μm; inset of b, c, e, and f 20 μm; g–p 100 μm; q 20 μm
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Fig4: Immunohistochemical analysis of cell migration-associated molecule expression in liver allograft endothelia. VCAM-1 and ICAM-1 expression was upregulated after LTx (a–e). Note reciprocal expression pattern for VCAM-1 and ICAM-1 at portal vein and sinusoidal endothelia, respectively (b versus e). VCAM-1 expression was slightly induced in the syngeneic graft (f). The expressions of P-selectin (g and l), PECAM-1 (h, m), VAP-1 (i, n) and tissue fibronectin (k, p, and q, arrowhead) was upregulated while sialyl-6-sulfo-Lewis X (j, o) declined at the PV endothelia after LTx. Scale bars: a, c, and d 200 μm; b, e, and f 100 μm; inset of b, c, e, and f 20 μm; g–p 100 μm; q 20 μm
Mentions: Next, we analyzed the expression of cell migration-associated molecules on the graft vascular endothelium by immunohistochemistry. Several cell migration-associated molecules were expressed constitutively and selectively on the PV endothelia in normal donor livers as shown in Table 1. From day 2 after LTx, vascular cell adhesion molecule-1 (VCAM-1) was preferentially induced on the portal but not sinusoidal endothelia and persisted thereafter (Fig. 4a–c). Occasionally, endothelia of the central vein were also partly positive for this molecule. Intercellular cell adhesion molecule-1 (ICAM-1) expression was also upregulated after LTx but was restricted to sinusoidal and hepatic vein endothelia (Fig. 4d, e). Weak expression of VCAM-1 in the PV (Fig. 4f) and of ICAM-1 on sinusoidal endothelia (not shown) was occasionally seen in the syngeneic graft on day 2. P-selectin, platelet/endothelial cell adhesion molecule (PECAM)-1, and vascular adhesion protein (VAP)-1 were constitutively expressed and slightly upregulated in the PV endothelia after LTx (Fig. 4g–i, l–n). In contrast, the expression of sialyl-6-sulfo Lewis X (CD15s), a ligand of selectins, was downregulated in the PV endothelia from day 2 after LTx (Fig. 4j–o). Tissue fibronectin (tFN), one of the ligands of α4β1 integrin, which is upregulated after LTx [12], was temporarily expressed in the PV endothelia from day 3 after LTx (Fig. 4k, p and q). E-selectin expression was unchanged, and neither mucosal vascular addressing cell adhesion molecule-1 (MAdCAM-1) nor ICAM-2 was detected on the surface of the graft vasculature (not shown). The spatio-temporal expression profile of these molecules is summarized in Table 1.Table 1

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.