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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.


Expression of chemokines and chemokine receptors expression after LTx. CXCR3 expression in liver perfusate at day 3 after LTx (a, b).  *p < 0.05. Note that high β1 integrin expression was preferentially induced in CXCR3+CD8 T cells after LTx (c, filled red) compared with control (c, filled gray). Preferential migration of CXCR3+ cells (blue) in portal tract in a day 3 allograft (d). Note attaching CXCR3+ cell in the PV endothelial wall (black arrowhead in inset). Some CXCR3+ cells were actively proliferating (white arrowhead in inset). Triple immunofluorescence staining with CXCL10 (red) and type IV collagen-like structure (white pseudocolor) with donor MHCII (e and g, green) or CD163 (f, green) in a day 3 allograft. Note co-localization of CXCL10 in donor MHCIIhigh cells in the portal tract (e) as well as CD163 in the sinusoid (f, white arrowhead). Also note the deposition of CXCL10 in the PV vessel wall (g, yellow arrowheads). Scale bars: d and e, 100 μm; inset of d and e, 20 μm; f, 50 μm; g, 20 μm; inset of g, 10 μm
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Fig6: Expression of chemokines and chemokine receptors expression after LTx. CXCR3 expression in liver perfusate at day 3 after LTx (a, b).  *p < 0.05. Note that high β1 integrin expression was preferentially induced in CXCR3+CD8 T cells after LTx (c, filled red) compared with control (c, filled gray). Preferential migration of CXCR3+ cells (blue) in portal tract in a day 3 allograft (d). Note attaching CXCR3+ cell in the PV endothelial wall (black arrowhead in inset). Some CXCR3+ cells were actively proliferating (white arrowhead in inset). Triple immunofluorescence staining with CXCL10 (red) and type IV collagen-like structure (white pseudocolor) with donor MHCII (e and g, green) or CD163 (f, green) in a day 3 allograft. Note co-localization of CXCL10 in donor MHCIIhigh cells in the portal tract (e) as well as CD163 in the sinusoid (f, white arrowhead). Also note the deposition of CXCL10 in the PV vessel wall (g, yellow arrowheads). Scale bars: d and e, 100 μm; inset of d and e, 20 μm; f, 50 μm; g, 20 μm; inset of g, 10 μm

Mentions: First, we investigated expression of chemokine receptors in activated T-cells in the perfusate. Th1-related chemokine receptor CXCR3 but not CCR5 (Fig. 6a, b) or CXCR6 (not shown) was significantly upregulated in the LTx group compared to controls. Notably more β1 integrinhigh T-cells were seen in the CXCR3+ population than in their CXCR3− counterparts in LTx group. In particular, CXCR3+CD8β+ T-cells upregulated β1 integrin, in which a proportion of β1 integrinhigh cells was 79.7 ± 5.8 % in the LTx liver perfusates compared to 42.0 ± 6.3 % in the controls. CXCR3+CD4+ T-cells, defined by CXCR3+TCRαβ+CD8β− population, constitutively expressed high levels of β1 integrin (Fig. 6c). Immunohistochemistry of day 3 graft livers also showed the expression of CXCR3 by migrated cells in the portal tract and those in the immediate vicinity of the PV endothelia (Fig. 6d). CCR9, known as a gut-homing molecule [13], was not detected (not shown).Fig. 6


Direct evidence for activated CD8+ T cell transmigration across portal vein endothelial cells in liver graft rejection
Expression of chemokines and chemokine receptors expression after LTx. CXCR3 expression in liver perfusate at day 3 after LTx (a, b).  *p < 0.05. Note that high β1 integrin expression was preferentially induced in CXCR3+CD8 T cells after LTx (c, filled red) compared with control (c, filled gray). Preferential migration of CXCR3+ cells (blue) in portal tract in a day 3 allograft (d). Note attaching CXCR3+ cell in the PV endothelial wall (black arrowhead in inset). Some CXCR3+ cells were actively proliferating (white arrowhead in inset). Triple immunofluorescence staining with CXCL10 (red) and type IV collagen-like structure (white pseudocolor) with donor MHCII (e and g, green) or CD163 (f, green) in a day 3 allograft. Note co-localization of CXCL10 in donor MHCIIhigh cells in the portal tract (e) as well as CD163 in the sinusoid (f, white arrowhead). Also note the deposition of CXCL10 in the PV vessel wall (g, yellow arrowheads). Scale bars: d and e, 100 μm; inset of d and e, 20 μm; f, 50 μm; g, 20 μm; inset of g, 10 μm
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Fig6: Expression of chemokines and chemokine receptors expression after LTx. CXCR3 expression in liver perfusate at day 3 after LTx (a, b).  *p < 0.05. Note that high β1 integrin expression was preferentially induced in CXCR3+CD8 T cells after LTx (c, filled red) compared with control (c, filled gray). Preferential migration of CXCR3+ cells (blue) in portal tract in a day 3 allograft (d). Note attaching CXCR3+ cell in the PV endothelial wall (black arrowhead in inset). Some CXCR3+ cells were actively proliferating (white arrowhead in inset). Triple immunofluorescence staining with CXCL10 (red) and type IV collagen-like structure (white pseudocolor) with donor MHCII (e and g, green) or CD163 (f, green) in a day 3 allograft. Note co-localization of CXCL10 in donor MHCIIhigh cells in the portal tract (e) as well as CD163 in the sinusoid (f, white arrowhead). Also note the deposition of CXCL10 in the PV vessel wall (g, yellow arrowheads). Scale bars: d and e, 100 μm; inset of d and e, 20 μm; f, 50 μm; g, 20 μm; inset of g, 10 μm
Mentions: First, we investigated expression of chemokine receptors in activated T-cells in the perfusate. Th1-related chemokine receptor CXCR3 but not CCR5 (Fig. 6a, b) or CXCR6 (not shown) was significantly upregulated in the LTx group compared to controls. Notably more β1 integrinhigh T-cells were seen in the CXCR3+ population than in their CXCR3− counterparts in LTx group. In particular, CXCR3+CD8β+ T-cells upregulated β1 integrin, in which a proportion of β1 integrinhigh cells was 79.7 ± 5.8 % in the LTx liver perfusates compared to 42.0 ± 6.3 % in the controls. CXCR3+CD4+ T-cells, defined by CXCR3+TCRαβ+CD8β− population, constitutively expressed high levels of β1 integrin (Fig. 6c). Immunohistochemistry of day 3 graft livers also showed the expression of CXCR3 by migrated cells in the portal tract and those in the immediate vicinity of the PV endothelia (Fig. 6d). CCR9, known as a gut-homing molecule [13], was not detected (not shown).Fig. 6

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 CD8&beta;+ 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&ndash; and upregulated &alpha;4&beta;1 or &alpha;L&beta;2 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 &alpha;4&beta;1 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.