It is currently accepted that tissue deposition of C4d, the final split product resulting from the proteolytic cleavage of complement element C4, is a marker of humoral alloreactivity (18
). In order to investigate the role of HLA antibody in complement-dependent HR of the liver allograft in ABO identical/compatible transplantation, we corroborated the tissue deposition of C4d with the concurrent detection of circulating DSA, a strategy currently in clinical use to diagnose AMRin other solid organ allografts.To our knowledge, except for a few case reports, no study has correlated the DSA detection by the sensitive and specific method of Luminex with C4d deposition in liver allografts undergoing acute and chronic rejection. Our subjects underwent clinically indicated liver biopsy to evaluate the cause of abnormal liver tests both in the early (<90 days) as well as the late posttransplantation period, and the study biopsies covered the severity spectrum of ACR, ductopenic rejection or no rejection, being therefore representative for the rejection types seen in our practice.
Methodologically, we performed for practical reasons immunostaining for C4d on paraffin embedded tissues, although some raised the possibility that this method may have lower sensitivity than staining of frozen tissue (28
No consensus has been developed regarding the C4d pattern of significance for detection of HR in liver allografts. Most studies () have reported C4d deposition in portal tracts, noticing immunolabeling of vascular structures (including endothelium of portal capillaries, veins, sinusoids, central veins and hepatic arteries), and portal stroma (20
). In our study the most common site of C4d deposition was the portal capillaries and nearby stroma, presumably due to leakage of C4d from the damaged endothelium, and covalent binding via the exposed thioester group to the closest protein or carbohydrate in the matrix near the site of complement activation and C4d formation (32
C4d immunolabeling patterns, detection methods and association with rejection in ABO compatible liver transplants reported in the literature
In our study, a heterogeneous C4d immunolabeling pattern was observed, and the diffuse portal C4d deposition had a statistically significant association with ACR. This is in line with other studies which have shown that the extensive, diffuse portal C4d deposition had clinical significance in terms of rejection and graft survival (20
), and analogous with demonstration of humoral alloreactivity seen in other allografts, where the diffuse C4d deposition and DSA positivity is required for the diagnosis of HR, with yet unclear clinical significance of lesser degree of C4d deposition (4
). We therefore considered the detection of circulating DSA with diffuse portal C4d deposition in combination with histological rejection an indicator of complement-dependent humoral alloreactivity specifically mediated by antibodies against donor HLA molecules.
Thirty percent of the cases falling in this category had also histological findings potentially characteristic of AMR, namely histological evidence of biliary outflow obstruction without radiological obstruction of the large bile ducts.
Sinusoidal endothelial C4d immunolabeling has been reported mostly in association with portal deposition (), with one group remarking on its absence (19
). However in a well-documented case of pure HLA-AMR a predominantly diffuse sinusoidal C4d deposition was observed by immunofluorescence using monoclonal antibodies (21
). We observed sinusoidal C4d immunolabeling almost exclusively in association with portal C4d staining. Hence, we could not determine the significance of isolated sinusoidal C4d deposition. Based on our findings, sinusoidal C4d staining seems to represent a more widespread C4d deposition extending beyond the portal tracts downstream into the sinusoids. As such it may add to the specificity of C4d staining. Had we taken into account only the cases with sinusoidal C4d deposition, we would have missed several cases of AMR, including one of severe acute ductopenia with initially only portal C4d deposition (case 14).
A great variability (8–100%) in C4d deposition in ACR of ABO compatible liver allografts has been reported (). Based on the presence of both diffuse portal C4d deposition and detection of circulating DSA we found that the frequency of complement-dependent humoral alloreactivity to donor HLA in ACR was 53.6%. Overall diffuse portal C4d irrespective of DSA status was present in 67.8% of ACR cases, close to figures reported by other of 67.7% (19
) and 69.2% (35
). While it is possible that the C4d deposition occurred in HLA-antibody negative patients as a result of complement activation by other types of donor-specific alloantibodies (36
) it should also be noted that three of the four cases of lone diffuse C4d positivity associated with ACR had biliary obstruction and cholangitis. In these cases the C4d might have resulted from complement activation by infection via the lectin or C-reactive pathways (37
). The detection of C4d should not however be invariably attributed to cholangitis when this infection is present. For example, case no. 27 had no C4 deposition in spite of having ischemic biliary strictures and severe bacterial cholangitis. Future prospective studies will allow consideration of other circumstances that might complicate C4d interpretation in liver allografts in the presence of inflammatory liver diseases shown to result in C4d deposition even in native livers (39
The portal C4d deposition is pathogenically concordant both with the localization of inflammatory infiltrate and the overexpression of HLA antigens in the structures in this region during ACR. Indeed in acute rejection class I antigens are increased, and class II antigens, especially DR, DP but also DQ, become overexpressed on endothelial cells, and bile ducts (41
). In our study antibodies against both HLA classes appeared involved in humoral alloreactivity, in conformity with other recent studies (14,31 and ).
Earlier studies have postulated a role for lymphocytotoxic antibodies against donor HLA in the pathogenesis of ductopenia (9
). Recent studies based on retrospective tissue immunolabeling demonstrated C4d deposition in chronic ductopenic rejection in 17% (45
) to 100% (29
) of cases. Seventy percent of the patients with ductopenia in our study had DSA, and 60% of ductopenia cases had both circulating DSA in association with diffuse portal C4d deposition, supporting a role for AMR in the pathogenesis of interlobular bile duct injury and loss. The resolution of cholestasis and ductopenia in association with reduction of C4d deposition only after decrease in circulating DSA with aggressive therapy specifically directed toward antibody removal further supports a role for DSA in the pathogenesis of ductopenia. Of particular importance is the risk of development of acute ductopenia as seen in this study, an operational distinction previously defined as an irreversible, rejection-related condition of acute vanishing bile duct syndrome occurring within 100 days after liver transplantation (47
). Recognition of the role of HLA-AMR in the pathogenesis of this rare but serious entity is essential in order to salvage the allograft by timely initiation of specific antibody-depleting therapy.
Corroborating our findings of significant portal C4d deposition in DSA positive cases with the elegant demonstration by morphometric studies of portal microvasculature destruction preceding the bile duct loss in the process of rejection (48
) we subscribe to the previously proposed mechanism in which the biliary tree may behave similarly to the kidney tubules because of their arterial only blood supply and conventional arteriolar and capillary plexus, the injury of which triggered by HLA antibodies could account for ductopenia (9
). Therefore the following chain of events seems to occur: formation of DSA-HLA complex on endothelial cells of the portal tract microvasculature triggers complement activation (evidenced by C4d deposition) and destruction of the portal microvasculature/capillaries (32
) branching off the communicating artery from which periductal vascular plexus arises (49
), resulting in ischemic bile duct injury and loss.
Humoral mechanisms of alloreactivity mediated by donor-specific HLA antibodies appear to frequently operate along with cellular mechanisms of rejection. This may explain the general benefit of including mycophenolic acid derivatives in the prevention and treatment of liver rejection, as it inhibits B-cell proliferation and antibody production. Evidence exists that the commonly used CNI tacrolimus and cyclosporine also affect antibody formation through interference with Th cells essential for the B cell-mediated immune responses (1
). This aspect helps explain the response in most cases of mixed cellular and HR of the liver to current immunosuppressant therapy directed primarily against T cell. (52
). Further studies are required to understand the level of immune dysregulation of alloantibody production responsible for the rejection refractoriness to the usual immunosuppressive therapy seen on occasion as progressive cholestasis due to ongoing bile duct damage and loss.
Our study is limited by the biases inherent to any retrospective survey. A selection bias is introduced by the non-consecutive nature of our cases. This bias is reduced by having most degrees of clinical and histological severity of rejection represented in patients with and without DSA, as well as cases without rejection. Due to the absence of protocol biopsies (which are not performed at our institution) and surveillance DSA determination, we are unable to determine the presence of subclinical AMR, as our subjects were selected to undergo liver biopsy indicated by liver dysfunction. Our study does not allow accurate gauging of the possible clinical significance of focal C4d deposition or of circulating DSA without associated C4 deposition. Because of the very limited number of follow-up biopsy with C4d staining, we can not provide data about the duration of C4d persistence, or the dynamics of DSA after rejection treatment and return of liver tests to baseline. As the C4d binds to the nearby proteins or carbohydrates covalently, it is expected that it takes the half-life of those substances, and hence to have a variable tissue persistence. Serial biopsies will be required to determine the dynamics of C4d deposition. Our study population is skewed toward patients with acute cellular and chronic ductopenic rejection. We believe that a properly resourced prospective study will be necessary to accurately assess the spectrum of humoral alloreactivity and to allow standardization of detection methodologies in liver transplant recipients, while limiting the confounding influence of biases that are inherent in a retrospective study.
In summary, it appears that humoral alloreactivity mediated by antibodies against donor HLA molecules is frequently intertwined with cellular mechanisms in the process of rejection of ABO identical/compatible liver allografts. Our study also supports the view that AMR in liver transplantation has a role in the destruction of the portal microvasculature leading to ischemic injury of the interlobular bile ducts, and ductopenia. This is particularly critical when it develops in the early posttransplant period as rapidly progressing cholestasis with a potentially inexorable course may endanger the liver allograft viability unless therapy specifically directed toward antibody depletion is initiated. An important conclusion of our study is that there is a need for prospective studies to correlate the detection of circulating DSA with C4d tissue deposition, histology and outcome of the liver allograft in order to advance our understanding of the significance of AMR after liver transplantation.