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Primary Biliary Cirrhosis: Cells, Sera, and Soluble Factors

      The information base on primary biliary cirrhosis (PBC) has dramatically improved during the past several years. Until 1985, PBC was known primarily as an idiopathic autoimmune disease mainly of female patients with antibodies directed against mitochondria.
      • Kaplan MM
      Primary biliary cirrhosis.
      The autoantibodies to mitochondria were poorly defined, were considered relatively nonspecific, and were associated with false-positive results. In addition, the nature of the mitochondrial autoantigens was unknown. Furthermore, a lengthy, clumsy, and confusing mitochondrial autoantigen nomenclature had been proposed.
      • Gershwin ME
      • Mackay IR
      • Sturgess A
      • Coppel RL
      Identification and specificity of a cDNA encoding the 70 kd mito-chondrial antigen recognized in primary biliary cirrhosis.
      The situation dramatically changed when a complementary DNA that expressed the major mitochondrial autoantigen of PBC was cloned, sequenced, and ultimately identified as the E2 subunit of the pyruvate dehydrogenase complex.
      • Gershwin ME
      • Mackay IR
      • Sturgess A
      • Coppel RL
      Identification and specificity of a cDNA encoding the 70 kd mito-chondrial antigen recognized in primary biliary cirrhosis.
      • Van de Water J
      • Fregeau D
      • Davis P
      • Ansari A
      • Danner D
      • Leung P
      • et al.
      Autoantibodies of primary biliary cirrhosis recognize dihydrolipoamide acetyltransferase and inhibit enzyme function.
      Shortly thereafter, the mitochondrial auto-antigens were identified as components of the 2-oxo-acid dehydrogenase complex; these features have been reviewed and discussed in detail elsewhere.
      • Leung PSC
      • Gershwin ME
      The molecular structure of autoantigens.
      This discovery alleviated much of the confusion about the nomenclature of the mitochondrial autoantigens. PBC is now one of the few autoimmune diseases in which the major autoantigens have been identified and sequenced. Additionally, reactive peptides and clones that encompass the major epitopes have been defined. Nonetheless, despite these serologic advances, major gaps in our knowledge remain.
      Many of the gaps in our understanding of the natural history and pathogenesis of PBC revolve around certain paradoxes of the disease. First, it is virtually the only autoimmune disease that has never been reported in childhood. Second, PBC increases in frequency with advancing age, and it can be found in otherwise asymptomatic elderly persons. Third, although associations with the major histocompatibility complex have been described, the genetic basis of PBC and the association of genetics and environment remain unknown. Fourth, some patients with histologically proven PBC do not have mitochondrial autoantibodies. Additionally, in PBC as in other autoimmune diseases, the autoantigen is found in every cell in the body; other diseases are tissue-specific conditions targeted to the biliary ductule epithelium. Finally, one should remember the importance of the cellular infiltrate in patients with PBC. Most likely, PBC is mediated by T cells, and the autoantibody response to mitochondrial antigens is a footprint whose importance, function, and relationship to disease may be entirely a secondary phenomenon.
      In this issue of the Mayo Clinic Proceedings (pages 1049 to 1055), Hashimoto and colleagues report an interesting immunohistochemical analysis of liver tissue from 20 patients with PBC. The purpose of the study was to identify the presence of activated T cells, interferon-γ-producing cells, and natural killer cells in portal tract infiltrates as well as in areas of piecemeal necrosis. The authors also examined the bile ducts for further corroboration of earlier findings
      • Ballardini G
      • Mirakian R
      • Bianchi FB
      • Pisi E
      • Doniach D
      • Bottazzo GF
      Aberrant expression of HLA-DR antigens on bileduct epithelium in primary biliary cirrhosis: relevance to pathogenesis.
      • Nakanuma Y
      • Kono N
      Expression of HLA-DR antigens on interlobular bile ducts in primary biliary cirrhosis and other hepatobiliary diseases: an immunohistochemical study.
      of class II human leukocyte (HLA) antigens on the bile duct epithelial cell surface. Two other liver diseases, primary sclerosing cholangitis and chronic active hepatitis, were included as disease controls.
      The results of the current study substantiated previous findings
      • Si L
      • Whiteside TL
      • Schade RR
      • Starzl TE
      • Van Thiel DH
      T-lymphocyte subsets in liver tissues of patients with primary biliary cirrhosis (PBC), patients with primary sclerosing cholangitis (PSC), and normal controls.
      • Krams SM
      • Van de Water J
      • Coppel RL
      • Esquivel C
      • Roberts J
      • Ansari A
      • et al.
      Analysis of hepatic T lymphocyte and immunoglobulin deposits in patients with primary biliary cirrhosis.
      of CD4-positive T cells in the portal spaces in PBC. CD8-positive cells were predominantly found in the areas of piecemeal necrosis. Also noted were CD22-positive cells (or B cells) intermixed with the CD3 cells in the portal tracts. An interesting finding was that few, if any, interferon-γ-producing cells were noted in the inflammatory infiltrates. Previously, several groups had proposed that the aberrant expression of class II HLA antigens on bile duct epithelial cells was the result of local inflammatory cell production of interferon-γ.
      • Skoskiewicz MJ
      • Colvin RB
      • Schneeberger EE
      • Russell PS
      Widespread and selective induction of major histocompat-ibility complex-determined antigens in vivo by yinterferon.
      On the basis of the data reported by Hashimoto and colleagues, this mechanism may not be responsible for class II HLA expression by the bile duct epithelial cells in PBC, primary sclerosing cholangitis, or chronic active hepatitis.
      This study raises several questions about the immunopathologic features of PBC and the two control diseases; several issues need to be addressed:
      • 1.
        If interferon-γ-producing cells are absent from bile duct lesions, what factors induce the presence of class II antigens on bile duct epithelium in all three liver diseases?
      • 2.
        Other immunologic factors should be considered when periductal inflammation in PBC is studied—for instance, what role, if any, do intercellular adhesion molecules, CD45R0 (activated T cells), CD22 cells, and immunoglobulin play in biliary destruction?
      • 3.
        Because all three bile duct diseases examined in this study had similar cell infiltrate populations, except for an increase in natural killer cells in primary sclerosing cholangitis, are the described cell populations due to a generic rather than a specific inflammatory response?.
      If interferon-γ-producing cells are absent from bile duct lesions, what factors induce the presence of class II antigens on bile duct epithelium in all three liver diseases? In several systems, investigators have shown that interferon-γ stimulates the expression of class II HLA antigens on epithelial cells in culture.
      • Vachier I
      • Godard P
      • Michel FB
      • Descomps B
      • Damon M
      Aberrant expression of HLA-DR antigens of the MHC class II on bronchial epithelial cells in asthmatic patients.
      • Baudouin C
      • Fredj-Reygrobellet D
      • Jambou D
      • Gastaud P
      • Lapalus P
      HLA DR and DQ expression on human retinal pigment epithelial cells in vitro.
      No such effect was noted with the application of interleukin 2, phorbol myristate acetate, fibroblast growth factor, insulin, or phytohemagglutinin-protein fraction. In the current Mayo study, Hashimoto and coworkers specifically attempted to determine whether interferon-γ-producing cells were present in bile duct lesions of patients with PBC, primary sclerosing cholangitis, and chronic active hepatitis. The authors failed to detect a substantial number of interferon-γ-producing cells by using immunohistochemical means. These findings are similar to recent results with use of molecular technology to analyze the messenger RNA levels of cytokines in the periductal lesions of patients with PBC.
      • Shindo M
      • Mullin GW
      • Bergasa NV
      • Jones EA
      • James SP
      Lack of cytokine mRNA expression in the liver of patients with primary biliary cirrhosis [abstract].
      The possibility exists that early events involving the stimulation of epithelial cell presentation of class II HLA antigens have occurred before the sampling times of stages II to IV used in these studies. This hypothesis would necessitate that the maintenance of class II HLA expression be controlled by some stimulation other than interferon-γ.
      To determine alternative factors involved in bile duct epithelial class II HLA expression entails considerable speculation. Griffiths and associates
      • Griffiths CEM
      • Voorhees JJ
      • Nickoloff BJ
      Characterization of intercellular adhesion molecule-1 and HLA-DR expression in normal and inflamed skin: modulation by recombinant gamma interferon and tumor necrosis factor.
      reported that the cytokine tumor necrosis factor can induce HLA-DR expression on epithelial cells. Interestingly, some bile acids have a direct inhibitory effect on release of tumor necrosis factor a from mononuclear cells. Because both PBC and primary sclerosing cholangitis are cholestatic disorders that result in the accumulation of bile acids, various microenvironments may be created by the bile acids and may thus influence cytokine production as the disease progresses.
      • Broome U
      • Hultcrantz R
      • Scheynius A
      Lack of concomitant expression of ICAM-1 and HLA-DR on bile duct cells from patients with primary sclerosing cholangitis and primary biliary cirrhosis.
      • Greve JW
      • Gouma DJ
      • Buurman WA
      Bile acids inhibit endotoxin-induced release of tumor necrosis factor by mono-cytes: an in vitro study.
      Therefore, the possibility exists that the initial cytokine and cellular profile is altered during the course of the disease, and what is seen at stages II to IV may, at least partially, be the cumulative result of the disease process.
      Other immunologic factors should be considered when periductal inflammation in PBC is studied—for instance, what role, if any, do intercellular adhesion molecules, CD45R0 (activated T cells), CD22 cells, and immunoglobulin play in biliary destruction? For occurrence of cell-mediated destruction of bile duct epithelium, lymphocytes must presumably adhere to the target cells. This process is accomplished by the integrin family of cell adhesion molecules that are constitutively expressed by leukocytes, fibroblasts, and endothelial cells. The leukocyte integrins, such as LFA-1 and VLA-4, have an important role in the immune response by selectively binding to their target ligands (ICAM-1, 2, and 3 for LFA-1 and VCAM-1 for VLA-4) at the site of inflammation.
      • Dougherty GJ
      • Murdoch S
      • Hogg N
      The function of human intercellular adhesion molecule-1 (ICAM-1) in the generation of an immune response.
      Thus, the role of adhesion molecules has been investigated in several autoimmune diseases, including both PBC and primary sclerosing cholangitis. Livers from patients with end-stage PBC and primary sclerosing cholangitis were found to have strong expression of ICAM-1 on both interlobular and proliferating bile ducts; the amount of ICAM-1 expression correlated with the degree of inflammation. Because this expression was much weaker in precirrhotic livers in patients with PBC, ICAM-1 may not be involved in initiation of the disease. Therefore, other adhesion molecules may be present on bile duct epithelium at different times during the course of disease, and these should be examined as well.
      • Broome U
      • Hultcrantz R
      • Scheynius A
      Lack of concomitant expression of ICAM-1 and HLA-DR on bile duct cells from patients with primary sclerosing cholangitis and primary biliary cirrhosis.
      • Adams DH
      • Hubscher SG
      • Shaw J
      • Johnson GD
      • Babbs C
      • Rothlein R
      • et al.
      Increased expression of intercellular adhesion molecule 1 on bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis.
      In the current study, Hashimoto and associates used anti-CD11b as a marker for suppressor T cells/monocytes and found few cells of this phenotype. Of note, the CD11b antigen is associated with the CD18 integrin heterodimer. As previously mentioned, investigators must consider that the cell populations involved in PBC are not static and likely change as the microenvironment changes during the disease process.
      Hashimoto and colleagues also discuss the discrepancy between their finding of few interleukin 2 receptor-positive, or activated, T cells in the periportal lesions and the previous frequent finding reported by Colucci and associates.
      • Colucci G
      • Schaffner F
      • Paronetto F
      In situ characterization of the cell-surface antigens of the mononuclear cell infiltrate and bile duct epithelium in primary biliary cirrhosis.
      An additional leukocyte antigen that could be used to examine the portal tract inflammatory response and T-cell activation more accurately is the leukocyte common antigen (LCA or CD45). This antigen has been associated with the transition from naive, unprimed T cells (CD45RA) to memory or activated T cells that are capable of responding to recall stimulation (CD45R0). Furthermore, this transition is thought to be accompanied by the coordinated up-regulation adhesion molecules, including LFA. Studies have shown, however, that the intensity of T-cell surface LFA-1 expression is not closely associated with the status of cell maturation but rather reflects the degree of cellular activation.
      • Hviid L
      • Ødum N
      • Theander TG
      The relation between T-cell expression of LFA-1 and immunological memory.
      The distinction between CD45RA- and CD45R0-positive subsets of T cells has proved extremely relevant in relationship to immunoactivation and immunopathologic changes. CD45R0-positive T cells have been shown to have a key role in immunoactivation and have been associated with the pathogenesis of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis.
      • Müller K
      • Bendtzen K
      Inhibition of human T lymphocyte proliferation and cytokine production by 1,25-dihydroxyvitamin D3: differential effects on CD45RA+ and CD45R0+cells.
      Finally, Hashimoto and colleagues found a significant number of CD22-positive cells in both PBC and chronic active hepatitis. Previous work by Krams and coworkers
      • Krams SM
      • Van de Water J
      • Coppel RL
      • Esquivel C
      • Roberts J
      • Ansari A
      • et al.
      Analysis of hepatic T lymphocyte and immunoglobulin deposits in patients with primary biliary cirrhosis.
      revealed not only a significant number of B cells in the portal tract but also the presence of IgM, IgG, and IgA both in the lymphoid infiltrate and on the surface of the bile duct epithelium. The role of B cells and immunoglobulins in the immunopathogenesis of PBC is unknown. One cannot dismiss the possibility, however, that the destruction of bile duct epithelium in PBC may be mediated by both T and B cells; thus, these cells should be investigated at both the functional and the phenotypic level.
      Because all three bile duct diseases examined in this study had similar cell infiltrate populations, except for an increase in natural killer cells in primary sclerosing cholangitis, are the described cell populations due to a generic rather than a specific inflammatory response? With use of the particular reagents described, the periductal cell populations apparently are similar in all three liver diseases analyzed. The only outstanding difference noted was an increase in the natural killer cell population in primary sclerosing cholangitis. The similarity of the remaining cell population suggests that, at the disease stages studied, a general rather than a specific immunopathologic response may be occurring. In all three groups, 90% of the patients had expression of HLA-DR on the bile duct epithelium. This finding indicates that some activation mechanism for class II expression is present in all three diseases. The difficulty of determining whether this expression is merely a response to immune injury and activation or has a role in the disease process itself remains in obtaining samples from tissue early in the disease process or, even better, during disease ontogeny.

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