Alloimmune activation enhances innate tissue inflammation/injury in a mouse model of liver ischemia/reperfusion injury

Abstract

The deleterious sensitization to donor MHC Ags represents one of the most challenging problems in clinical organ transplantation. Although the role of effector/memory T cells in the rejection cascade has been extensively studied, it remains unknown whether and how these 'Ag-specific' cells influence host innate immunity, such as tissue inflammation associated with ischemia and reperfusion injury (IRI). In this study, we analyzed how allogeneic skin transplant (Tx) affected the sequel of host's own liver damage induced by partial warm ischemia and reperfusion. Our data clearly showed that allo-Tx recipients had increased inflammatory response against IR insult in their native livers, as evidenced by significantly more severe hepatocelluar damage, compared with syngeneic Tx recipient controls, and determined by serum ALT levels, liver histology (Suzuki's score) and intrahepatic proinflammatory gene inductions (TNF-alpha, IL-1beta and CXCL10). The CD4 T cells, but neither CD8 nor NK cells, mediated the detrimental effect of allo-Ag sensitization in liver IRI. Furthermore, CD154, but not IFN-gamma, was the key mechanism in allo-Tx recipients to facilitate IR-triggered liver damage. These results provide new evidence that alloreactive CD4 T cells are capable of enhancing innate tissue inflammation and organ injury via an Ag-nonspecific CD154-dependent but IFN-gamma independent mechanism.

Figure 1

Alloimmune activation enhances liver IRI. (a). WT C57BL/6 mice were either left untreated (naïve), or were challenged with a syngeneic (Syn) or MHC-fully-mismatched Balb/c (Allo) skin grafts 10 days prior to being subjected to liver IR, as described in Material and Methods. Liver injury was evaluated at 6 h and 24 h post-reperfusion by measuring sALT levels (n=11–14/group). Liver tissue was harvested at 6 h and 24 h post reperfusion for histological and molecular analyses. Representative liver H/E sections (100x) from each experimental group are shown (b), as well as their average Suzuki’s scores (c). Liver expression of TNF-α, CXCL10, and IL-1β genes was determined by target gene/HPRT ratios and measured by qRT-PCR (d, n=3–4/group).

Figure 1

Alloimmune activation enhances liver IRI. (a). WT C57BL/6 mice were either left untreated (naïve), or were challenged with a syngeneic (Syn) or MHC-fully-mismatched Balb/c (Allo) skin grafts 10 days prior to being subjected to liver IR, as described in Material and Methods. Liver injury was evaluated at 6 h and 24 h post-reperfusion by measuring sALT levels (n=11–14/group). Liver tissue was harvested at 6 h and 24 h post reperfusion for histological and molecular analyses. Representative liver H/E sections (100x) from each experimental group are shown (b), as well as their average Suzuki’s scores (c). Liver expression of TNF-α, CXCL10, and IL-1β genes was determined by target gene/HPRT ratios and measured by qRT-PCR (d, n=3–4/group).

Figure 2

Effector/memory T cells in sensitized recipients. T cell activation in naïve, syn- or allo-skin transplanted recipients was evaluated by FACS analysis of spleen or liver-infiltrating lymphocytes, as described Material and Methods. CD4+ or CD8+ lymphocytes were gated and further analyzed for their CD62L and CXCR3 expression. Effector (or effector memory) T cells were defined by the frequency of CXCR3⁺CD62L^low subpopulation (circled). Representative density plots of effector CD4 or CD8 T cells in spleens and livers are shown (a), and their average percentages in each experimental group are plotted (b). N=3/group

Figure 2

Effector/memory T cells in sensitized recipients. T cell activation in naïve, syn- or allo-skin transplanted recipients was evaluated by FACS analysis of spleen or liver-infiltrating lymphocytes, as described Material and Methods. CD4+ or CD8+ lymphocytes were gated and further analyzed for their CD62L and CXCR3 expression. Effector (or effector memory) T cells were defined by the frequency of CXCR3⁺CD62L^low subpopulation (circled). Representative density plots of effector CD4 or CD8 T cells in spleens and livers are shown (a), and their average percentages in each experimental group are plotted (b). N=3/group

Figure 3

CD4 T cells mediate the enhanced liver IRI in sensitized recipients. C57BL/6 mice were either left untreated (naïve), or transplanted with a MHC-fully-mismatched Balb/c (Allo) skin graft. At day 9 post skin challenge, recipient mice were treated with CD4, CD8, or NK cell-depleting Ab, as described in Material and Methods. At day 10, these mice were subjected to liver partial warm ischemia (90 min), followed by evaluation of the hepatocellular damage (sALT levels) at 6 h post-reperfusion (a, n=6–8/group). Liver tissue samples were also harvested, and representative H/E sections (100x) are shown (b). Liver expression of TNF-α, CXCL10, and IL-1β, as determined by target gene/HPRT ratios, and measured by qRT-PCR (c, n=3–4/group).

Figure 4

Allogeneic-Ag activated CD4 T cells recreated liver IRI in nude mice. Nude mice were either untreated (Gr#1) or reconstituted with purified syngeneic CD4 T cells from either syn- (Gr#2) or allo-geneic (Gr#3) skin Tx recipients, as described in the material and methods. CD4 T cells were detected by FACS at similar levels in both groups of reconstituted nude mice (a). These mice were then subjected to liver partial warm ischemia (90 min), followed by measurement of sALT levels at 6 h post-reperfusion (b). Representative liver H/E sections (100x) from each experimental group are shown (c) and Suzuki scores for each groups were plotted (d). Liver expression of TNF-α, CXCL10, and IL-1β, measured by target gene/HPRT ratios, were determined by qRT-PCR (e). n=4/group.

Figure 4

Allogeneic-Ag activated CD4 T cells recreated liver IRI in nude mice. Nude mice were either untreated (Gr#1) or reconstituted with purified syngeneic CD4 T cells from either syn- (Gr#2) or allo-geneic (Gr#3) skin Tx recipients, as described in the material and methods. CD4 T cells were detected by FACS at similar levels in both groups of reconstituted nude mice (a). These mice were then subjected to liver partial warm ischemia (90 min), followed by measurement of sALT levels at 6 h post-reperfusion (b). Representative liver H/E sections (100x) from each experimental group are shown (c) and Suzuki scores for each groups were plotted (d). Liver expression of TNF-α, CXCL10, and IL-1β, measured by target gene/HPRT ratios, were determined by qRT-PCR (e). n=4/group.

Figure 5

CD154 signaling is critical for alloimmune-mediated liver IRI. C57BL/6 mice were transplanted with MHC-fully-mismatched Balb/c (Allo) skin grafts. At day 10, recipient mice were treated with either control Ig, or anti-CD154 or anti–IFN-γ Ab, as described in Material and Methods. These mice were then subjected to liver partial warm ischemia (90 min), followed by measurement of sALT levels at 6 h post-reperfusion (a, n=5–8/group). Representative liver H/E sections (100x) from each experimental group are shown (b). Liver expression of TNF-α, CXCL10, and IL-1β, determined by target gene/HPRT ratios, and measured by qRT-PCR (c, n=3–4/group).