AUTOANTIBODIES

AMA were described for the first time in patients with PBC in the 1960s and continue to be regarded as the most sensitive and specific immunologic hallmark of the disease. AMA are directed to the E2 component of the pyruvate dehydrogenase complex (PDC-E2), the E2 unit of the branched-chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), and the E2 subunit of the 2-oxo-glutarate dehydrogenase complex (OGDC-E2). Other AMA recognize the E1a subunit of PDC (PDC E1a) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) of PDC. These molecules all are located on the inner mitochondrial membranes. At least one of these components usually reacts with AMA in a patient with PBC. The most frequent antigen against which AMA are directed is PDC-E2; PDC-E2–reacting antibodies are present in 90% to 95% of PBC sera.

The mechanisms by which AMA directed to proteins located on the inner surface of mitochondrial membranes develop are unknown; PDC-E2 or a cross-reactive molecule is overexpressed on biliary epithelial cells in PBC, predominantly at the luminal domain, and PDC-E2–specific CD4⁺ T cells are present in portal inflammatory infiltrates of affected persons. Although AMA are predominantly of the IgG1 and IgG3 classes, most patients who have PBC exhibit polyclonal elevation of serum IgM levels; the IgM is not directed at mitochondrial or nuclear antigens. This phenomenon is suggestive of polyclonal activation of the B-cell compartment with an associated failure of isotype switching, representing aberrant B-cell activation.¹¹ AMA do not appear to be cytotoxic: (1) They persist after liver transplantation without evidence of disease recurrence; (2) disease severity is unrelated to antibody titer; (3) they are not always present in PBC; and (4) they develop in animal models after the injection of recombinant PDC-E2 protein, but without resulting bile duct destruction or inflammation. Further, the different types and numbers of mitochondrial antigens recognized by Western immunoblot analysis at the time of the patient’s presentation is independent of the stage of the liver disease and not associated with specific clinical, biochemical, histologic, and immunologic features or with the Mayo risk score (see later).¹²

Antinuclear antibodies (ANA) are present in nearly one half of patients with PBC and in up to 85% of patients with AMA-negative PBC (see later). The most relevant immunofluorescent reactivities of ANA in patients with PBC are anti-multiple nuclear dots antibodies ([anti-MND], with the molecular target being a 100-kd soluble protein called Sp100), anticentromere antibodies, and antinuclear envelope antibodies. The immunofluorescence pattern of the antinuclear envelope antibodies is characterized as being rim-like and membranous; its molecular targets are structural components of the nuclear pore complex, such as gp210 and nucleoprotein p62, and of the nuclear membrane, such as lamin B receptors. Antibodies against the nuclear pore protein gp210 (anti-gp210) are found in 25% of patients with AMA-positive PBC and in up to 50% of those with AMA-negative PBC. ANA with the MND and rim-like and membranous patterns, which are relatively rare or absent in normal and pathologic controls, are strongly associated with PBC and can be considered to be surrogate markers of PBC in AMA-negative patients.^13–15 The specificity of anti-gp210 for PBC when detected by immunoblotting is greater than 99%, whereas antibodies to p62 (anti-p62) are found in approximately 25% of patients and are highly specific for PBC. Anti-p62 antibodies seem to be mutually exclusive with anti-gp210 antibodies. Further, anti-gp210 and possibly anti-p62 also offer prognostic information in that they seem to be associated with aggressive disease with a poor prognosis.^13,16,17 In Japanese patients with PBC, anticentromere antibodies are associated with the development of portal hypertension.¹⁸

GENETIC FACTORS

The occurrence of PBC in relatives of affected persons plus abnormalities of cell-mediated immunity in first-degree relatives of patients with PBC suggests a genetic association. This association is further supported by the finding that PBC exhibits a higher concordance rate in monozygotic than dizygotic twin pairs, suggesting a genetic component to disease susceptibility and expression of the susceptibility through genes that regulate the immune response.¹⁹ Many of the familial risk and genetic studies of PBC have failed to distinguish, however, between true genetic risk and shared environmental exposure.

Although no link between PBC and a specific human leukocyte antigen (HLA) class I phenotype has been found, HLA class II molecules may contribute to the development of this condition. The HLA associations with PBC detected most commonly have been with the DRB1*0801 allele in European and North American white populations and DRB1*0803 in Japanese populations.^20,21 Although the DRB1*08 allele seems to impart a significant risk for PBC, with odds ratios of 3 or higher in white and Japanese populations, a study from China reported that the frequencies of the DRB1*0701 and DRB1*03 alleles were increased significantly in patients with PBC compared with controls, with no difference in the frequencies of the DRB1*08 allele.²² A few class II HLA alleles demonstrate protective associations against PBC. These alleles include DQA1*0102 in United States and Japanese studies and DQB1*0602 in a United States study. More recently, DRB*13 was found to be protective against PBC in patients from the United Kingdom and Italy, and DRB1*11 was protective in patients from Italy but not those from the United Kingdom.^20,23 An association with HLA class III genes, which code for complement components C2 and C4, cytokines, and complement factor B, has been studied less extensively. Earlier studies reported an increased frequency of haplotype C4B2 and haplotype C4A*Q0 in patients with PBC. Smaller, older studies of the association between PBC and alleles affecting the expression of tumor necrosis factor-α have been reported, but an association remains unresolved.

MOLECULAR MIMICRY

Molecular mimicry between host autoantigens and unrelated exogenous proteins is one of the hypotheses to explain how autoantibodies to self-proteins arise, break tolerance, and lead to autoimmune disease. Molecular mimicry of an extrinsic protein produced by an infectious agent has long been suggested as a possible initiating event in PBC. Infectious agents incriminated in the immune response in PBC include various bacteria and viruses, most recently Chlamydia pneumoniae,²⁴ Novospingobium aromaticivorans,²⁵ and human betaretrovirus.²⁶ Microorganisms produce a multitude of foreign antigens that collectively constitute the major determinants recognized by the immune system. These antigens potentially include a variety of carbohydrates, lipids, and proteins that can be recognized by specific receptors on inflammatory cells. In PBC, PDC-E2 appears to be an ideal candidate for foreign antigens to mimic. PDC-E2, particularly its inner lipoyl domain, is highly conserved among bacteria, yeasts, and mammals. Autoimmune phenomena in PBC could result from peptides that mimic T-cell epitopes of microbial proteins and that are derived from, and presented by, abnormally expressed HLA class II molecules. Molecular mimicry has been invoked to explain the breaking of tolerance against mitochondrial antigens. Definitive evidence for this theory is still lacking, however.

XENOBIOTICS AND OTHER IMPLICATED AGENTS

Xenobiotics are foreign compounds that may alter self-proteins by inducing a change in the molecular structure of the native protein sufficient to induce an immune response. The immune response may then result in the recognition of both the modified and the native proteins.²⁷ The continued presence of the self-protein may perpetuate the immune response initiated by the xenobiotic-induced adduct, thereby leading to chronic autoimmunity. Because many xenobiotics are metabolized in the liver, the potential for liver-specific alteration of proteins is substantial. To address the hypothesis that PBC is induced by xenobiotic exposure, Long and colleagues²⁸ synthesized the inner lipoylated domain of PDC-E2, replaced the lipoic acid moiety with synthetic structures, and quantified the reactivity of these structures with sera from patients with PBC. AMA from all patients reacted more strongly to 3 of the 18 modified organic autoepitopes than to the native domain. Defective sulfoxidation of certain compounds, such as bile acids, estrogen, or drugs, and selenium deficiency have been proposed as underlying mechanisms that may lead to this process. These hypotheses remain unproved.

ASYMPTOMATIC DISEASE

Widespread use of screening laboratory tests has led to diagnosis of PBC at an asymptomatic stage in up to 60% of patients with this condition. Such patients are found incidentally to have an elevated serum alkaline phosphatase level and AMA during routine health evaluations or during investigation of an unrelated complaint, such as a clinical manifestation of an autoimmune disease known to be associated with PBC. Some asymptomatic persons with AMA and normal results of liver biochemical tests are found to have features on liver biopsy specimens that are diagnostic of or consistent with PBC; symptoms, signs, and laboratory evidence of chronic cholestasis eventually develop in these persons.

SYMPTOMATIC DISEASE

The typical patient with symptomatic disease (Table 89-1) is a middle-aged woman with a complaint of fatigue or pruritus. Other symptoms include right upper quadrant abdominal pain, anorexia, and jaundice. Fatigue, although relatively nonspecific, is considered to be the most disabling symptom by many patients, and it worsens in some patients as the disease progresses.^29,30 Fatigue in patients with PBC does not correlate with several markers of disease severity, or with the patient’s age or thyroid status, but does correlate with sleep disturbance and depression.²⁹ In a four-year follow-up study, the severity of fatigue was quite stable overall; only liver transplant recipients experienced a significant improvement in fatigue.³¹ In that study, fatigue was also found on multivariate analysis to be an independent predictor of mortality, particularly cardiac death.³¹

Table 89-1 Symptoms and Signs of Primary Biliary Cirrhosis at Presentation

Pruritus may occur at any point, early or late, in the course of the disease, or intermittently throughout the course. Pruritus generally is intermittent during the day and is most troublesome in the evening and at night. Pruritus often resolves as the disease progresses, but in some patients, severe, intractable pruritus can develop in earlier stages of the disease and may require liver transplantation for effective management. In a population-based study of 770 patients with PBC from England, the cumulative risk of developing pruritus was 19%, 45%, and 57% at 1, 5, and 10 years, respectively.³⁰

Most patients with PBC do not have jaundice at the time of diagnosis. Jaundice occurs later in the course of the disease and usually is persistent and associated with a worse prognosis. Symptoms also may relate to fat-soluble vitamin deficiency, bone pain with or without spontaneous fractures, or an associated autoimmune disease that may occur in patients with PBC (Table 89-2). Symptoms and signs of advanced liver disease, such as ascites, bleeding from gastroesophageal varices, and encephalopathy, usually occur late in the course of PBC.

Table 89-2 Diseases Associated with Primary Biliary Cirrhosis

CREST, calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia.

On physical examination, the most common signs are skin hyperpigmentation (caused by deposition of melanin), hepatosplenomegaly, xanthelasma, and, in more advanced disease, jaundice. Symptoms appear to be less frequent in men than in women, and autoimmune manifestations, especially Sjögren’s syndrome, also are less frequent in men. Otherwise, PBC is identical clinically in men and in women.

ASSOCIATED DISEASES

Many of the diseases found frequently in patients with PBC (see Table 89-2) are thought to be related to disturbances in immune mechanisms. These associated disorders include Sjögren’s syndrome (characterized by dry eyes [keratoconjunctivitis sicca] and dry mouth), scleroderma and its variants, rheumatoid arthritis, some cutaneous disorders, renal tubular acidosis, and thyroiditis.

The frequency of malignancy is increased in patients with PBC. An increased risk of breast cancer in women with PBC was found in earlier studies but was not confirmed by subsequent, larger studies. Although hepatocellular carcinoma occurs in only 1% to 2% of patients with PBC, the risk is much higher than that in the general population. Gallstones can be found in up to one third of patients with PBC, but inflammatory bowel disease and interstitial pulmonary fibrosis are rare.

BIOCHEMICAL FEATURES

Liver biochemical test results show a cholestatic picture. Almost all patients have increased serum levels of alkaline phosphatase (three to four times the upper limit of normal) and gamma glutamyl transpeptidase. Serum aminotransferase (aspartate aminotransferase [AST], alanine aminotransferase [ALT]) levels are mildly elevated (usually less than three times normal); marked elevations (more than five times normal) are distinctly unusual and may suggest PBC-autoimmune hepatitis overlap syndrome (see Chapter 88) or coexisting viral hepatitis. Serum bilirubin levels usually are normal in early stages and increase slowly over the course of the disease; levels ultimately may exceed 20 mg/dL. A high serum bilirubin level, low serum albumin, and prolonged prothrombin time indicate a poor prognosis and advanced disease. Serum immunoglobulin levels, especially IgM, are increased, as are serum levels of bile acids, in particular cholic and chenodeoxycholic acids, and cholesterol.

SEROLOGIC DIAGNOSIS

Indirect immunofluorescence, immunoblotting, and enzyme-linked immunosorbent assay (ELISA) can detect AMA. Indirect immunofluorescence is by far the most commonly used serologic test and detects AMA in 90% to 95% of patients with PBC; however, indirect immunofluorescence testing requires interpretation by a skilled observer, and the result may be interpreted erroneously as negative for AMA in some patients with PBC. Immunoblotting and ELISA have sensitivity and specificity rates higher than 95% for the detection of AMA in PBC and can detect AMA in patients with PBC who are AMA negative by direct immunofluorescence testing. Other autoantibodies found in patients with PBC include rheumatoid factor (70%), smooth muscle antibodies (66%), antinuclear antibodies (50%), and antithyroid (antimicrosomal, antithyroglobulin) antibodies (41%).

HISTOPATHOLOGIC FEATURES

The initial lesion on a liver biopsy specimen in PBC (Figs. 89-1 and 89-2A and B) is damage to epithelial cells of the small bile ducts. The most important and only diagnostic clue in many cases is ductopenia, defined as the absence of interlobular bile ducts in greater than 50% of portal tracts. The florid duct lesion, in which the epithelium of the interlobular and segmental bile ducts degenerates segmentally, with formation of poorly defined, noncaseating epithelioid granulomas, is nearly diagnostic of PBC but is found in a relatively small number of cases, mainly in early stages.

Figure 89-1. Schematic representation of the staging system of primary biliary cirrhosis (Ludwig’s classification). The left side of the schematic shows five portal tracts surrounding a central vein at each stage. The right side shows a larger single portal tract at each stage (the bile ductule is blue). In stage 1 the inflammation is confined to the portal space, focused on the bile duct. In stage 2 the inflammation extends into the hepatic parenchyma (interface hepatitis or piecemeal necrosis). In stage 3, fibrosis is present; in stage 4, cirrhosis is present.

Figure 89-2. A, Photomicrograph of stage 2 primary biliary cirrhosis (PBC). Mononuclear inflammatory cells expand the portal tracts with some disruption of the limiting plates (interface hepatitis). The bile ducts are surrounded by inflammatory cells, and no fibrosis is evident. (Hematoxylin and eosin, ×100.) B, Stage 4 PBC. Cirrhosis, with areas of fibrosis surrounding the hepatic parenchyma, is present. A dense mononuclear inflammatory infiltrate is still seen in the portal tract, with interface hepatitis. (Hematoxylin and eosin, ×100.)

The two most popular histologic staging systems are those proposed by Ludwig and colleagues and Scheuer, which classify the disease in four stages. Both systems describe progressive pathologic changes, beginning initially in the portal areas surrounding the bile ducts and culminating in cirrhosis. Ludwig stage 1 disease is characterized by inflammatory destruction of the intrahepatic septal and interlobular bile ducts that range up to 100 µm in diameter. These lesions often are focal and described as florid duct lesions, characterized by marked inflammation and necrosis around a bile duct. The portal tracts usually are expanded by lymphocytes, with only sparse neutrophils or eosinophils seen. In stage 2 disease (see Fig. 89-2A), the inflammation extends from the portal tract into the hepatic parenchyma, a lesion called interface hepatitis, or formerly, piecemeal necrosis. Destruction of bile ducts with proliferation of bile ductules can be seen. Stage 3 disease is characterized by scarring and fibrosis. Lymphocytic involvement of the portal and periportal areas, as well as the hepatic parenchyma, can be seen, but the hallmark of this stage is the presence of fibrosis without regenerative (or regenerating) nodules. Stage 4 disease is characterized by cirrhosis with fibrous septa and regenerative nodules (see Fig. 89-2B).

Most patients with PBC demonstrate progression of the liver disease; a few patients have a prolonged course of histologic stability, and only rare patients have sustained regression. A time course Markov model has been used to describe the rate of histologic progression over time (Table 89-3).