The CD154-CD40 T-cell co-stimulation pathway in liver ischemia and reperfusion inflammatory responses

Abstract

Background: Ischemia-reperfusion (I/R) injury is a prime antigen-independent inflammatory factor in the dysfunction of liver transplants. The precise contribution of T cells in the mechanism of I/R injury remains to be elucidated. As the CD154-CD40 co-stimulation pathway provides essential second signal in the initiation and maintenance of T-cell-dependent immune responses, this study was designed to assess the role of CD154 signaling in the pathophysiology of liver I/R injury.

Methods: A mouse model of partial 90-min warm hepatic ischemia followed by 6 hr of reperfusion was used. Three animal groups were studied: (1) wild-type (WT) mice treated with Ad-(-gal versus Ad-CD40 immunoglobulin; (2) untreated WT versus CD154 (MR1) monoclonal antibody-treated WT mice; and (3) untreated WT versus CD154 knockout mice.

Results: The disruption of CD154 signaling in all three animal groups ameliorated otherwise fulminant liver injury, as evidenced by depressed serum glutamic oxaloacetic transaminase levels, compared with controls. These beneficial effects were accompanied by depressed hepatic T-cell sequestration, local decrease of vascular endothelial growth factor expression, inhibition of tumor necrosis factor-(and T-helper type 1 cytokine production, and induction of antiapoptotic (Bcl-2/Bcl-xl) but depression of proapoptotic (caspase-3) proteins.

Conclusions: By using in parallel a gene therapy approach, pharmacologic blockade, and genetically targeted mice, these findings document the benefits of disrupting CD154 to selectively modulate inflammatory responses in liver I/R injury. This study reinforces the key role of CD154-CD40 T-cell co-stimulation in the pathophysiology of liver I/R injury.

FIGURE 1

SGOT levels in mice that underwent 90 min of hepatic warm ischemia followed by 6 hr of reperfusion. Groups included sham-operated controls; T-cell–competent untreated WT mice; WT mice that were pretreated with Ad-CD40Ig, Ad-(-gal (2.5 × 10⁹ plaque-forming units at day −2), or CD154 mAb (MR1; 0.25 mg at days −2 and −1); and CD154-deficient (KO) mice. There were four to six animals in each group. Means and SD are shown.

FIGURE 2

T-cell (CD3⁺) infiltration in murine livers undergoing I/R injury with or without CD154-CD40 co-stimulation blockade. Data are representative of three recipients per group (immunoperoxidase labeling of cryostat sections, hematoxylin counterstained; magnification ×400). For details, see Materials and Methods and Figure 1.

FIGURE 3

Reverse-transcriptase PCR-assisted detection of mRNA coding for VEGF in hepatic ischemic lobes at 6 hr of reperfusion after 90 min of warm ischemia. For details, see Materials and Methods and Figure 1. There were four to six animals in each group. Means and SD are shown.

FIGURE 4

Reverse-transcriptase PCR-assisted detection of mRNA coding for TNF-( and Th1 (IL-2 and IFN-() cytokines in hepatic ischemic lobes at 6 hr of reperfusion after 90 min of warm ischemia. For details, see Materials and Methods and Figure 1. There were four to six animals in each group. Means and SD are shown.

FIGURE 5

Western blot analysis of antiapoptotic (Bcl-2/Bcl-xl) gene products in mouse livers after 90 min of warm ischemia followed by 6 hr of reperfusion. The protein expression was probed by using antibodies against mouse Bcl-2 (A), Bcl-xl (B), and caspase-3 (C). (lane 1) Sham-operated control; (lane 2) WT mice; (lane 3) WT mice treated with Ad-(-gal; (lane 4) WT mice treated with Ad-CD40Ig; (lane 5) WT mice treated with CD154 mAb; (lane 6) CD154 KO mice. Data shown are representative of three separate experiments.