In the present study, we have demonstrated that a significantly higher percentage of PSC patients have autoantibodies to surface antigens expressed on BECs compared with PBC, AIH, and normal individuals. In addition, we have shown that these autoantibodies induced increased expression and production of CD44 and IL-6, respectively, on cultured BEC. Thus for the first time we report the presence and functional capacity of autoantibodies in sera of PSC patients to surface antigens expressed on the clinically relevant target cells of destruction, namely BECs.
Initially, the main immunoglobulin class of anti-BEC antibodies detected in PSC sera was IgM. The presence of IgG anti-BEC autoantibodies however was detected only after isolation of the IgG fraction from the sera of these patients. It is likely that higher levels of the IgM pentamer immunoglobulin may sterically hinder or mask the binding of divalent IgG antibodies. Such findings have also been reported in other studies.19
In addition, as stated in the introduction, approximately half of all PSC patients have an abnormal increase in IgM levels thus adding to the masking of IgG antibody binding. Thus anti-BEC autoantibodies may be both IgM and IgG. Interestingly, in some PSC patients with end stage disease, predominantly IgG anti-BECs antibodies were found. In addition, the presence of IgG antibodies reflects the presence of cellular sensitisation.
Another interesting finding was the significant association of production of these autoantibodies with the HLA haplotype −B8, −DR3, or −DR2 found in PSC patients. Classic (type 1) AIH is also associated with the −DR3 allele but in this study only one AIH patient with this allele had anti-BEC antibodies. Neither sex nor age could account for this difference. Thus a heterogenous population of PSC patients may exist in whom different mechanisms may be involved in the pathogenesis of PSC depending on the presence or absence of anti-BEC antibodies.
Most PSC sera reacted with only cytokine stimulated BECs. This implies that a clinically important target(s) for antibody binding is probably induced or alternatively requires modification resulting in binding of antibodies.
Studies on the functional capacity of anti-BEC antibodies showed that not all anti-BEC antibodies could lyse BECs. A few patients with high titre anti-BEC antibodies had complement fixing capacity and were found to cause low level lysis of BECs. However, this did not correlate with any clinical parameter. An important effect of anti-BEC antibodies was induction of increased expression of CD44 on BECs by the Ig fractions isolated from PSC patients. Cruickshank et al
have reported that normal bile ducts do not express CD44 but increased expression of CD44 on BECs of PSC and some PBC livers was observed.20
In our study, expression of CD44 was not observed in freshly isolated BECs but purified cultured BECs (after passage 1) expressed CD44. This expression was not altered by treatment with TNF-α or IFN-γ, a finding similar to that reported by Cruickshank and colleagues.20
Thus actively proliferating cells express CD44. Induction of CD44 expression on BECs by anti-BEC autoantibodies has important implications—for example, these antibodies may facilitate the recruitment of memory T cells (via CD44) and activate other cellular mechanisms leading to the destruction of bile ducts in these patients. The multifunctional properties of CD4421–24
may in addition have other important clinical implications in the inflammatory process involved in PSC. Some of the possible functions for CD44 in diseased livers have been discussed by Cruickshank and colleagues.20
Induction of high levels of the proinflammatory cytokine IL-6 by anti-BECs autoantibodies further indicates their importance in the pathogenesis of these diseases. It has been reported that under conditions of inflammation or stress in vivo, BECs produce, secrete, and respond to IL-6.25
IL-6 induces BEC proliferation in vitro26, 27
and suppresses BEC apoptosis.28
This cytokine is markedly elevated in the bile of patients with cholangitis29
and serum of patients with cholangiocarcinoma.30
IL-6 has been suggested to exert a profibrogenic and mitoinhibitory effect on the development of cirrhosis.31–35
Thus the fibro-obliterative inflammation of both the intra- and extrahepatic biliary tree seen in PSC may be a consequence of persistent IL-6 production contributing to uncontrolled proliferation and fibrosis.
One of the primary roles of IL-6 in vivo is as the major cytokine that initiates the hepatic acute phase response.36
In addition, IL-6 is well established as a late stage differentiation factor for B cell to plasma cell transition,37
enhancing immunoglobulin production and augmenting secondary antibody responses to antigens in vivo.38
In PSC, a significant increase in the number of B lymphocytes which correlates with histological stage and serum levels of immunoglobulin and bilirubin has been reported.39
Our data suggest that anti-BEC autoantibodies transform BECs into an inflammatory phenotype. It is important to mention that recombinant IL-6 alone did not induce expression of CD44, a finding also shown by others,40
indicating that it is the anti-BEC antibodies that are responsible for induction of CD44 expression.
Taken together, the in vitro data suggest that a proinflammatory loop exists between the binding of autoantibodies to BECs and possible recruitment of mononuclear leucocytes via CD44 as well as sustained production of autoantibodies by B cells via IL-6. This may represent a coordinated inflammatory response that implicates both the cellular and humoral arm of the immune response in the pathogenesis of PSC. As a significantly high proportion of PSC patients have anti-BECs antibodies compared with PBC and AIH patients, it is tempting to speculate that probably antibodies play a more significant role in the pathogenesis of PSC.
The anti-BEC antibodies from PSC patients detected a specific band of 40 kDa not detected in the other patient groups. Similarly, 31 and 33 kDa bands were detected using sera from PBC patients not detected in the other patient groups. Several reasons may account for the increased intensity of bands obtained in western blots using PBC sera: (a) abundance of protein(s) expressed on a cell surface; (b) titre of antibodies; (c) affinity of antibodies (low titre antibodies may have high affinity); and (d) nature of the antigen(s) detected (if expression of certain antigens is dependent on structural/chemical conformation, the use of SDS may affect binding). However, further work is needed to identify the proteins (using the technology of proteomics) detected by anti-BEC antibodies in the sera of PSC and PBC patients. Such studies are currently being performed at our centre. Results from such studies will add to the knowledge of possible therapeutic interventions that may be used in the treatment of these patients.
No correlation between the presence of these antibodies and any clinical parameter was found in PSC patients. However, a significantly high proportion of PBC patients with end stage disease had anti-BEC antibodies. Thus these antibodies may be associated with progression of disease in these patients.
Even though in vivo expression of CD44 and IL-6 has been demonstrated on bile ducts of PSC and PBC patients, the mechanism for this increased expression is not known. In this study, we showed that one mechanism may be due to cross linking/binding of cell surface expressed specific protein/proteins by anti-BEC autoantibodies which may induce an intracellular signal(s) that upregulates expression of several molecules, among them CD44 and IL-6. In the past, the importance and contribution of autoantibodies in inflammatory processes involved in autoimmune liver diseases has been underestimated. However, using the clinically relevant target cells of destruction in PSC and PBC, we have shown that anti-BEC autoantibodies in these patients may have an important role in the pathogenesis of these diseases.