Apotope Of Human Intrahepatic Billary Epithelial Cells And The Billary-Specific Damage In Primary Billary Cirrhosis

Apoptosis, the major mechanism of programmed cell death, is essential in regulating and maintaining tissue growth as well as homeostasis. There are 50 to 70 billion cells die each day due to apoptosis in an average human adult. Clearance of apoptotic cells is a highly regulated process which is indispensable in preventing the outflow of intracellular content and limits the immunological responses against generated antigens. Apoptotic cells are normally cleared after engulfment by phagocytes followed by an anti-inflammatory response. The formation of apoptotic bodies (ABs) is essential during apoptosis to limit the escape of intracellular content and preclude any ensuing immunological responses against intracellular autoantigens with inflammatory reactions. Nevertheless, ABs can in some circumstances constitute be a major source of immunogens in autoimmune diseases that involve the targeting of ubiquitous autoantigens. Indeed, recent data have demonstrated that autoantigens found within ABs are critical in the presentation of antigens, the activation of innate immunity and the regulation of cytokine secretion by macrophages. ABs have been also reported as B cell autoantigens .We and others have described the post-apoptosis newly created antigens as apotope that comes from the word apoptosis and epitope.Primary biliary cirrhosis (PBC) is a typical organ-specific autoimmune disease results from a multilineage T and B cell response against an immunodominant mitochondrial autoantigen, identified as the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) and the E2 subunit two other 2-oxo acid dehydrogenase enzymes, the oxo-glutarate dehydrogenase complex (OGDC-E2) and the branched chain 2-oxo acid dehydrogenase complex (BCOADC-E2).PBC mainly affect woman over their 40s, and is characterized by the progressive lost of intrahepatic small bile duct, followed by the intrahepatic cholestasis and eventually liver cirrhosis and functional failure. A major unanswered question in the pathogenesis of PBC is why small bile ducts are selectively damaged when mitochondria are found in nearly all nucleated cell types. To address this issue, we have herein proposed the following hypothesis that HiBEC has post apoptosis protein clearance defect that result in the specific retain of PDC-E2 as well as other PBC-specific autoantigens in their ABs. These antigen-containing AB will be phagocytosis by professional or non-professional phagocytes, further, the constant leakage of intact cellular components may cause antigen accumulation in the vicinity of BECs and critically challenge the balance between homeostasis and autoimmunity, thus providing a structural basis for the eventual loss of tolerance in PBC. In addition, there is still little known about if the non-immune"neighbors"cells also play a role in this clearance of apoptotic cells, as the atypical distribution of PDC-E2 on the surface of BEC in patients has been described, we have a good reason to question that if the surface PDC-E2 is translocated from the ABs.Section I. The establishment of primary cultures of HiBECs and controls cell lines and apoptosis inductionHuman intrahepatic biliary epithelial cells (HiBECs), human bronchial epithelial cells (BrEPCs), and human mammary epithelial cells (MaEPCs) and human keratinocytes were cultured in sterile medium supplemented with 2% fetal bovine serum (FBS), epithelial cell growth supplement (ScienCell, San Diego, CA), and 1% penicillin in cell culture flasks coated with poly-L-lysine (Sigma-Aldrich, St Louis, MO). HiBECs were characterized using our standardized immunofluorescence method employing antibodies to cytokeratin 18, cytokeratin 19, and vimentin, which stained >90% of the cells. All experiments were performed between cell passage 2 and 5. Cells were cultured at 37°C in a humidified 5% CO2 incubator.Apoptosis in 4 cell lines were induced by three different methods. We first induce apoptosis with 1 mM sodium glycochenodeoxycholate (GCDC) for 4 hours at 37°C. Apoptosis was also induced by UVB irradiation (1650 J/m2-2200J/m2) followed by incubation in fresh medium for 6 hours, as well as by adding anti-Fas antibodies at 1μg/ml to the culture medium for 16 hours with the confirmation of surface expression of Fas in all cell lines. The GCDC effectively induced apoptosis in all cell lines,(more than 90% in HiBEC and 40%-50% in other 3 cell lines). The UVB irradiation results in 15-25% apoptosis in various cell lines whereas the anti-Fas antibodies only yield a ratio of apoptosis less than 5% in all cell lines.The supernatants were then passed through a 1.2μm nonpyrogenic hydrophilic syringe filter. After centrifugation at 100,000g for 45 minutes, the pellets containing apoptotic bodies were collected.Section II. Identification of apotopes of HiBECIn this section, we detected the presence of PDC-E2, OGDC-E2 and BCOADC-E2 ,three well defined PBC-specific autoantigens, and 4 proteins from mitochondrial inner membrane, DECR1, UQCRC2, COX-IV and ATPB, as well as 4 nucleus proteins,SSA,SSB,Sp100 and Gp210, that are associated with PBC by western blot or confocal microscope in ABs form 4 cell lines. The results showed that PDC-E2 and OGDC-E2 were specifically detected in ABs from HiBECs but not in ABs of the three other epithelial cell lines. DECR1, a 36-kDa subunit-sized protein of the mitochondrial matrix, was also present intact in ABs from HiBEC but not the other epithelial cells. Among the other proteins we examined, BCOADC-E2 and UQCRC2 were detected intact in ABs from HiBEC, BrEpC and MaEPC but not keratinocytes. SSA/Ro was detected in ABs from HiBEC and BrEpC whereas SSB/La was detected only in ABs from BrEpC. ATPB, COX-IV and Sp100 were not found in apoptotic bodies from any of the cell types examined. These data indicate that HiBEC is unique with respect to the preservation of mitochondrial autoantigens of PBC after apoptosis.Our data suggest that the defect of cellular protein PAD is not unique to HiBECs. We found several intact autoantigens in ABs of different epithelial cells, implying human epithelial cells variably process their apoptotic leftovers due to factors yet to be determined. Thus, all 3 mitochondrial 2-oxo acid dehydrogenase complexes, i.e. PDC-E2, OGDC-E2 and BCOADC-E2 that are autoantigens in PBC can be traced to HiBEC ABs. These findings highlight the involvement of inappropriate PAD as the source of autoantigens and perhaps in the pathogenesis of biliary-selective damage in PBC.Section III. Identification of apotopes of HiBECIn this section, we demonstrated the autoantibodies against the apotopes we previously identified in patient with PBC.Serum samples were obtained from human subjects diagnosed with PBC (n=114), systemic lupus erythematosus (SLE; n=23), primary sclerosing cholangitis (PSC; n=22) or unaffected controls (n=31). Patients with PBC and the three control groups were matched by gender and age. The 114 PBC patients include 108 females and 6 males. Ninety-five patients had serum antimitochondrial antibodies (AMA) while 19 were AMA negative.In all the 95 AMA positive PBC serum samples, 100% showed anti-PDC-E2 antibodies, 19 showed anti-OGDC-E2 antibodies and 25 showed anti-BCOADC-E2 antibodies. In addition, we also detected antibodies against DECR1 in 3 patients with PBC who also had serum antibodies against PDC-E2 .There was no reactivity of sera from AMA negative patients against any of the candidiate mitochondrial proteins studied herein.Section IV. The immune response of HiBEC after phagocytosis HiBEC-derived ABs We have previously reported that ABs from HiBEC were pathogenic when uptaken by macrophages of patients with PBC in the presence of antimitochondrial antibody (AMA).Besides, the surface staining of PDC-E2 in HiBEC of patients with PBC has been described. We therefore tried to answer the following questions in this section 1.) Weather HiBEC harbors the ability to process ABs from their apoptotic peers 2.) If HiBEC are involved in the local apoptotic clearance, does the immunogenic ABs raise any impact to their immunological behavior and possible influence to pathogenesis of PBC. Our data first demonstrated that HiBEC expresses a variety of phagocytosis-related surface receptors, including CD51, CD61, CD93 and PSR. In addition, HiBEC does has potent ability to uptake Abs, when coculture with fluoresce stained HiBEC-derived ABs, 34% of the recipient HiBECs showed fluoresce signal indicating the participate of apoptosis clearance, this observation was further confirmed by confocal microscope. Further, after the engulfment of ABs, the supernatants were then measured for the concentration of inflammatory cytokines and chemokines. Compare to the medium, we did not find any significant cytokine alteration, whereas 2 chemokines, CXCL-8 and CCL-2, were found to be increased by about 3-fold in the supernatant of HiBEC feed with ABs. The global profile of chemokines expression of AB-treated HiBEC was thus performed using the Human Chemokines & Receptors PCR Array. The data showed that HiBEC significantly upregulated the expression a variety of CCL family chemokines including CCL8, CCL16, CCL7 and CCL13.