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. 2006 Mar 21;103(12):4598-603.
doi: 10.1073/pnas.0600499103. Epub 2006 Mar 13.

Kupffer cell-dependent TNF-alpha signaling mediates injury in the arterialized small-for-size liver transplantation in the mouse

Yinghua Tian  1 , Wolfram Jochum, Panco Georgiev, Wolfgang Moritz, Rolf Graf, Pierre-Alain Clavien
Affiliations

Kupffer cell-dependent TNF-alpha signaling mediates injury in the arterialized small-for-size liver transplantation in the mouse

Yinghua Tian et al. Proc Natl Acad Sci U S A. .

Abstract

Implantation of small liver grafts causes liver injury and defective regeneration leading to graft failure. We investigated whether Kupffer cell-dependent TNF-alpha signaling contributes to this poor outcome. Partial 30% liver transplantation was performed in C57BL/6 wild-type mice (control group), and in three groups with down-regulation of the TNF-alpha pathway: (i) TNF receptor 1 knockout [TNFR-1(-/-)] mice, and mice pretreated with (ii) gadolinium chloride or (iii) pentoxifylline (PTX). Fifty-percent partial liver transplantation, a model associated with full recovery, and transplantation in IL-6 knockout [IL-6(-/-)] mice were performed in some experiments. Graft injury, regeneration, portal flow, liver microcirculation, leukocyte adhesion, and animal survival were assessed. Animal survival rates were 14% in the control group vs. 43% in the gadolinium chloride group, 57% for the TNFR-1(-/-) group, and 86% in the PTX group (P < 0.001). Markers of liver injury were reduced in all treated groups when compared with controls. Each treated group disclosed better portal flow and sinusoid perfusion, decreased leukocyte adherence, particularly in the PTX group. Liver regeneration occurred only in the treated groups. IL-6 and IL-10 levels were dramatically up-regulated (50x) in the PTX group, and at lower levels in other experimental groups. The protective effect of PTX was lost in IL-6(-/-) mice and protection was restored by a single dose of r-IL-6. In conclusion, interruption of TNF-alpha signaling or depletion of Kupffer cells improves survival after 30% liver transplantation, reduces liver injury, and enhances regeneration. The superior effects of PTX are mediated by IL-6.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Phagocytic activity after 30% OLT. To assess KC activity, the nonadherent latex particles were observed for 5 min in sinusoidal vein after injection and expressed as percentage of total latex particle. Untreated controls (■) showed very high phagocytic activities comparable with sham-operated animals (▵). GdCl3 (○) and PTX (formula image) pretreated animals had significantly reduced phagocytic activities compared with untreated controls (∗, P = 0.034 and P = 0.043, respectively). TNFR-1(−/−) (▾) mice showed comparable phagocytic activity as untreated controls (mean ± SD, n = 5 in each group).
Fig. 2.
Fig. 2.
Liver injury after transplantation. Serum AST levels were determined as markers of liver injury 2 days after surgery. Levels reached almost 2,500 units/liter in recipients of a 30% partial liver graft (untreated). In contrast, each strategy targeting on TNF-α signaling [PTX, TNFR-1(−/−)] or impairment of KC (GdCl3) disclosed lower AST levels, P < 0.02, for PTX and TNFR-1(−/−). Values for PTX and TNFR-1(−/−) were comparable with those observed after 50% partial OLT (mean ± SD, n = 5 in each group; ∗, P < 0.05 compared with untreated controls).
Fig. 3.
Fig. 3.
Histological analysis of graft tissue after transplantation. Hematoxylin/eosin staining of partial OLT grafts was performed 2 days after transplantation. In contrast to untreated 30% partial OLT grafts, which revealed diffuse microvesicular steatosis, only mild macrovesicular steatosis was found in 30% partial OLT grafts after treatment with PTX and GaCl3 and in TNFR-1(−/−) grafts. (Original magnification: ×200.)
Fig. 4.
Fig. 4.
Animal survival after transplantation. Kaplan–Meier analysis showed that recipients transplanted with a 30% partial graft (30% untreated controls) had a dramatically poorer survival (14%) compared with those receiving a 50% partial OLT graft (100% survival, P = 0.003). The strategies modulating TNF-α signals and KC activity had different effects on animal survival. Animals transplanted with a 30% partial graft and pretreated with PTX had a 86% long-term survival (P = 0.005 versus untreated controls), whereas TNFR-1(−/−) and GdCl3 pretreated animals had improved survival of 57% and 43% (P = 0.025 and P = 0.049 versus untreated controls, respectively; n = 7 in each group).
Fig. 5.
Fig. 5.
Portal flow after transplantation. Portal flow was measured 10 min, 3 h, and 2 days after partial OLT. In recipients transplanted with 30% partial grafts (■), portal flow decreased to 60% of baseline value by 10 min of reperfusion, and subsequently to 33% of baseline value by 2 days. Comparable initial post transplantation drops occurred in PTX (formula image), GdCl3 (○), and TNFR-1(−/−) (▾) groups, but then values remained stable or slightly improved over the subsequent 2 days. Fifty-percent OLT (□) showed a similar pattern. Sham-operated mice (▵) showed an initial drop in portal flow to ≈80% of baseline values without further changes (mean ± SD, n = 5 in each group; ∗, P < 0.05 when compared with the three protective strategies).
Fig. 6.
Fig. 6.
IVFM after 30% OLT. Microcirculatory changes 3 h after transplantation indicating nonperfused sinusoids (A) and leukocyte adhesion (B). In TNFR-1(−/−) mice, PTX and GdCl3 pretreated animals disclosed only slightly higher values than sham-operated animals (∗, P < 0.01 compared with untreated controls). Approximately 900 adherent leukocytes per mm2 were observed in untreated 30% grafts (B), whereas this number was halved in PTX, TNFR-1(−/−), and GdCl3 groups. Sham-operated animals presented fewer than 10 adherent leukocytes per mm2 (mean ± SD, n = 5 in each group).
Fig. 7.
Fig. 7.
Liver regeneration in partial OLT grafts. Quantitation (A) of hepatocyte proliferation analyzed in partial OLT grafts 2 days after transplantation using immunostaining for Ki-67 protein (B). Ki-67-positive hepatocytes were absent in untreated 30% partial OLT grafts but were numerous in 30% partial OLT grafts after treatment with PTX or GdCl3, and in TNFR-1(−/−) grafts, suggesting rescue of impaired regeneration (original magnification: ×200) (∗, P < 0.01).
Fig. 8.
Fig. 8.
Cytokine expression levels after 30% OLT. Transcript levels of protective cytokines IL-6 (dark-gray bars) and IL-10 (light-gray bars) 1 h after transplantation. PTX-treated animals showed dramatically increased IL-6 transcript compared with untreated controls (∗, P < 0.01). IL-10 mRNA levels in PTX-pretreated animals were significantly elevated compared with untreated controls (∗, P < 0.047; mean ± SD, n = 5 in each group).
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