In this study we show that Fas/FasL and CD40/CD40L are increased in the human liver allograft during rejection, and provide evidence that these pathways cooperate in mediating hepatocyte death in chronic allograft rejection. We confirm the ability of Fas activation to induce apoptosis of human hepatocytes in vitro (9
) and in addition show for the first time that engagement of hepatocyte CD40 by Ab or soluble CD40L is an equally potent inducer of apoptosis. The ability of CD40 ligation to induce hepatocyte death was surprising, given that the CD40 cytoplasmic tail does not contain a conventional death domain, and suggested to us that an indirect mechanism might be operating. We propose that this mechanism involves activation of Fas by autocrine or paracrine FasL because (a) CD40 ligation induces hepatocyte FasL expression, thereby providing the ligand to activate Fas on autologous or adjacent hepatocytes, and (b) CD40-induced apoptosis was blocked by a neutralizing anti-FasL Ab. Thus CD40 engagement can trigger Fas-dependent apoptosis of human hepatocytes and the HepG2 cell line, suggesting that this is an important mechanism for apoptotic death in cells of hepatocyte lineage.
The Fas pathway has been implicated in hepatocyte apoptosis in several diseases (12
). In normal human liver we observed only weak Fas expression on hepatocytes and no FasL. Findings were similar in biopsies from stable transplants but not in those from patients with allograft rejection in whom striking upregulation of both Fas and FasL was observed. CD40 was not detected in normal liver, but in chronic allograft rejection it was upregulated on hepatic endothelium, bile ducts, and particularly on centrilobular hepatocytes in areas of hepatocyte loss. That hepatocyte CD40 expression was only seen in chronic rejection whereas Fas was detected in both acute and chronic rejection suggests that the coexpression of Fas and CD40 might be a factor in determining progression from acute to chronic rejection.
Fas and FasL expression was also detected on the portal infiltrate in acute rejection, reflecting the presence of activated lymphocytes within the graft. Although increased Fas expression by hepatocytes could increase their susceptibility to cytolysis by FasL-bearing CTLs, hepatocyte damage is not a conspicuous feature of acute rejection and our previous studies showed little hepatocyte apoptosis in acute rejection (3
). Coexpression of CD40 and Fas on centrilobular hepatocytes was only seen in chronic rejection, and it is these patients who develop hepatocyte apoptosis. This suggests that these molecules cooperate to cause target cell damage and thus determine the severity of allograft rejection. The present study provides a molecular mechanism to explain this. Isolated primary human hepatocytes expressed both CD40 and Fas in culture, allowing us to determine the consequences of engaging hepatocyte cell surface Fas and CD40. The expression of CD40 and Fas on human hepatocytes in culture is at odds with the apparent absence of staining in normal tissue. This anomaly is probably due to activation induced by the isolation and culture procedures, because previous studies have shown CD40 upregulation on cultured epithelial cell lines (16
Cross-linking of hepatocyte Fas in vitro induced a high level of apoptosis, suggesting that FasL-bearing cells could promote hepatocyte apoptosis in chronic rejection. However, we detected relatively few FasL+
lymphocytes in chronic rejection despite many apoptotic hepatocytes, which led us to look for an alternative source of FasL. Under some circumstances hepatocytes express FasL which can cause Fas-mediated suicide or fratricide (14
), and we thought a similar mechanism could be involved in chronic rejection where centrilobular hepatocytes coexpress Fas and FasL. Moreover, activation of hepatocytes in vitro with cross-linking Ab resulted in an increase in hepatocyte cell surface FasL. IL-1 also upregulated cell surface FasL (Fig. a) and induced apoptosis in primary human hepatocytes (data not shown), and this has been previously reported in other types of epithelial cells (26
). Centrilobular hepatocytes in chronic rejection also stained strongly for CD40, and cross-linking of CD40 caused hepatocyte apoptosis that was comparable to that seen with direct Fas activation (Fig. ). Because the Fas pathway is known to be a potent inducer of hepatocyte apoptosis (9
), we investigated the consequences of CD40 ligation on FasL expression in isolated human hepatocytes.
Engagement of CD40 led to increased expression of cell surface FasL and to induction of apoptosis that was comparable to that induced by Fas cross-linking. Furthermore, CD40-induced apoptosis could be prevented by a neutralizing Ab to FasL, providing evidence of a direct link between CD40 and Fas in the regulation of apoptosis. The ability of hepatocytes to induce FasL-mediated apoptosis after engagement of CD40 suggests that CD40 promotes apoptosis via autocrine or fratricrine activation of Fas. It is notable that CD40 and Fas expression on centrilobular hepatocytes was seen in chronic rejection but not in severe acute rejection where the cells expressed Fas but not CD40. Hepatocyte apoptosis is not a conspicuous feature of acute rejection, and it is possible that Fas expression alone does not provide a potent enough signal.
Thus, CD40 may be a critical factor in the amplification of hepatocyte apoptosis in chronic rejection, and we subsequently looked for a cellular source of CD40L. Hepatocytes did not express CD40L protein or mRNA in vitro even after cytokine stimulation, and immunostaining of hepatocytes in tissue sections was consistently negative. However, CD40L was detected on CD68+
macrophages/ Kupffer cells in the centrilobular areas. In contrast very few infiltrating CD3+
T cells expressed CD40L. Thus, CD40L/ CD68+
macrophages could provide the ligand for activation of hepatocyte CD40 in chronic rejection. This model, in which continuing hepatocyte damage is promoted by activated macrophages rather than by cytolytic T cells, could explain why centrilobular hepatocyte dropout continues in chronic rejection in the absence of a marked lymphocytic infiltrate, and why chronic rejection often fails to respond to antilymphocyte therapy (1
In severe acute rejection CD40 was detected strongly on graft endothelium, whereas it was not detected in patients with mild self-limiting rejection, suggesting that CD40/ CD40L might also be involved in amplifying and maintaining lymphocyte recruitment during graft rejection. In support of this, recent studies have shown that ligation of CD40 on cultured endothelial cells results in increased expression of adhesion molecules, including vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1 ), that are expressed at high levels on endothelium in graft rejection. Thus, the induction and maintenance of these molecules might be a consequence of CD40 ligation on graft endothelium (27
Until now, little was known about the expression and function of CD40 in human liver. Our studies demonstrate a novel mechanism by which CD40L-bearing cells can amplify hepatocyte apoptosis through a Fas-dependent mechanism. A similar mechanism will likely apply to other diseases in which hepatocytes are destroyed by Fas activation, and in support of this we have recently detected CD40 on hepatocytes in viral hepatitis and alcoholic liver disease (12–15; Afford, S.C., P.L. Shields, and D.H. Adams, unpublished observations). We thus propose that this model is a mechanism of liver damage in a broad range of inflammatory liver diseases.