Clin. exp. Immunol. (1978) 34, 19-27.
Circulating IgG complexes in primary biliary cirrhosis. A serial study in forty patients followed for two years R. C. GUPTA, E. R. DICKSON, F. C. MCDUFFIE & A. H. BAGGENSTOSS Departments of Internal Medicine, Immunology and Pathology and Anatomy, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55901, USA
(Received 6 March 1978)
Several antibodies are present in sera of patients with primary biliary cirrhosis (PBC). We have looked for evidence of antigen-antibody complexes in sera of PBC assuming that some of the antibodies may circulate complexed with an antigen. The Raji cell radioimmunoassay, which determines complement-bound immune complexes, was used to determine the levels of such complexes in serial samples of sera from forty patients with PBC followed for 2 years. Twentyfour patients (60%) were found to have significantly elevated levels of circulating complexes. In the majority they were detected from the beginning of the study and the high levels persisted. In seven patients whose sera initially had normal levels of complexes, the levels increased to become abnormal during the following year. These complexes sedimented at > 19S in the majority of patients studied. The mean level of C3 but not C4 was lower in patients with elevated complexes than in those with normal complexes. A significant correlation was observed between the presence of elevated complexes and the severity of the inflammatory cell infiltrate surrounding intrahepatic portal tracts and serum IgG and IgM levels. There was also a significant correlation with titres of antimitochondrial antibody, but not with the histological stage of disease or with the collagen and copper content of the liver. Although the method of detection of immune complexes is indirect and the antigen is unknown, the presence of such high levels of complexes suggests a possible role of immune complexes in the pathogenesis of PBC. INTRODUCTION
Primary biliary cirrhosis (PBC) is a chronic, slowly progressive disease characterized by the nonsuppurative destruction of the intrahepatic bile ducts. The primary aetiological agents remain unknown. The disease occurs predominantly in women and has been associated with diseases of altered immune responsiveness, particularly scleroderma, Sjbgren's syndrome, and autoimmune thyroiditis (Golding et al., 1970; Sherlock & Scheuer, 1973). Some patients have a clinical syndrome resembling rheumatoid arthritis and several circulating autoantibodies have been described in these patients. Antimitochondrial antibodies (AMA) are present in the serum of about 90 to 95% of PBC patients (Klatskin & Kantor, 1972; Doniach, 1972; Sherlock & Scheuer, 1973) that react with the inner membrane of mitochondria and fix complement (Berg et al., 1969). However, no correlation has been found between the presence of titre of this antibody and the stage or severity of the disease. Other antibodies described in a few patients are against ductular cell cytoplasm (Paronetto, Schaffner & Popper, 1967), bile canaliculi (MacSween et al., 1973), and nuclei (Holborrow et al., 1963). Lymphocytotoxicity for autologous hepatocytes has been observed in some patients, especially those with florid lesions of the ducts and ductular proliferation (Geubel et al., 1976). These findings suggest the possibility of a pathogenesis related to iltered immune mechanisms. Correspondence: Dr R. C. Gupta, Assistant Professor of Medicine, Division of Rheumatic Diseases, University of :olorado Medical Center, Denver, Colorado 80262, USA. 0099-9104/78/0100-0019$02.00 (© 1978 Blackwell Scientific Publications
Gutpta et al.
The abnormal antibodies present in sera of patients who have PBC may be related to the disease and thus may circulate complexed with an antigen (an immune complex). Therefore, we looked for evidence of immune complexes in the sera ofpatients with PBC by the Raji cell radioimmunoassay. We found elevated levels of complexes in the sera of 60% of patients with PBC followed for 2 years, and a close relationship between elevated levels of complexes and the degree of inflammation of the intrahepatic cholangioles and titres of AMA and serum C3 (inverse correlation). The size of the complexes present has been found to be greater than 19S.
MATERIALS AND METHODS Patients. Patients with PBC in this study were selected on the basis of having all of the following: (1) evidence ofcontinuous liver disease based on history or biochemical abnormalities for longer than 3 months; (2) elevation of serum alkaline phosphatase to greater than three times the normal; (3) a positive mitochondrial antibody determination and (4) a pre-treatment liver biopsy characteristic of or compatible with the diagnosis of PBC (Baggenstoss et al., 1964). Patients were excluded from the study if they had a history suggesting chronic inflammatory bowel disease, malignancy during the past 10 years, use of a known cholestatic drug during the preceding 6 months, chronic alcoholism, extrahepatic biliary obstruction, or clinical and laboratory features consistent with the syndrome ofchronic active liver disease (Soloway et al., 1972). Collection of serum samples. Peripheral blood drawn from each patient, initially and at 6 month intervals, was allowed to clot at room temperature for 2 to 3 hr. Serum was separated and stored at -20'C in aliquots. All sera were thawed just before quantification of complexes and the test was performed in batches of forty to sixty samples each time. We found that freezing and thawing did not affect levels of complexes in the sera. In addition, samples of fresh sera from fifteen patients with PBC were examined for the presence of complexes. Method of immune complex detection. The method of quantifying immune complexes by Raji cell radioimmunoassay was that of Theofilopoulos, Wilson & Dixon (1976) which we have recently modified (Gupta et al., 1978). Briefly, 2 x 108 Raji cells in 50 jl RPMI-1640 tissue culture medium were reacted with 25 jl of a 1: 4 dilution in saline of the serum to be tested. The test sera was prepared by adding 300 Jl of saline and 10 ld of fresh normal human serum (NHS), as a source of complement, to 100 pl of patients' sera. After an incubation period of 45 min at 370C, with gentle shaking, the cells were washed three times with RPMI-1640. They were then reacted for 30 min at 40C, with continuous gentle shaking, with an optimum amount of a 1 : 2 dilution of 125I goat antihuman IgG in phosphate-buffered saline containing 1% bovine serum albumin (PBS-BSA). Subsequently, the cells were washed three times with PBS-BSA and radioactivity in the cell pellet was determined in a gamma counter. The amount of complexes present was determined by reference to a standard curve on which the amount of uptake of 125I goat antihuman IgG by cells was plotted against the amount of heat-aggregated human IgG (AHG) added to normal serum and then assayed as above. AHG was prepared by heating 7S human IgG in a water bath at 630C for 30 min. The amount of complexes in each serum tested was expressed in terms of pg equivalent AHG/ml serum Antimitochondrial antibodies (AMA). All tests for AMA were carried out on fresh serum samples from patients. Unfixed sections of rat kidney cut at 5 jim in a cryostat were air-dried on slides and overlaid with a 1 : 5 dilution of test serum. After incubation at 370C for 30 min, the sections were washed three times in 0-01M phosphate-buffered saline, pH 7 5, and then treated with fluorescein-labelled goat antihuman globulin (Meloy Laboratories, Inc., Springfield, Virginia, USA) and examined with a Leitz Ortholux immunofluorescent microscope using filter K530. Liver biopsies. An initial biopsy and subsequent yearly liver biopsies were performed in each patient. The histological picture in these specimens was interpreted by Dr A. H. Baggenstoss without knowledge of the clinical or laboratory data. The histological features were: (1) the stage of the disease: stage I, typical septal and intralobular duct lesions; stage II, ductal proliferation; stage III, periportal and septal fibrosis and stage IV, cirrhotic stage; (2) periportal inflammation graded as 1 (mild) to 3 (severe) on the basis of the degree and nature of the inflammatory cell infiltrate; (3) the amount of collagen and (4) the amount of hepatic copper (Baggenstoss et al., 1964). Serum immunoglobulins. IgG, IgM, and IgA levels were determined by a modification of the autoanalyser method described by Markowitz & Tschida (1972). Complement. C3 and C4 levels were determined by a radial immunodiffusion technique (Mancini, Carbonara & Heremans, 1965) on plates containing agar mixed with monospecific antiserum (Meloy Laboratories, Inc.,, Springfield, Virginia, USA). Sucrose density gradient studies. Density gradient ultracentrifugation was carried out in 12 x 75 mm tubes on 0 5 ml samples layered onto a 5-30% sucrose gradient in 0-1 M Tris, 0-2 mI NaCl, pH 8-0 buffer. Centrifugation was carried out at 283,000 g (at the bottom of the tube) for 12 hr at 40C in an SW 283 rotor in an International B-60 ultracentrifuge. All samples were run in duplicate. The gradient tubes were punctured after centrifugation and eighteen fractions (about 0-65 ml each) were collected. The fractions were tested for complement-fixing activity and for the presence of complexes. The complement-fixing activity was tested by a microtitre system (Hunder, Mullen & McDuffie, 1974). One drop (0.025 ml) of sample was added to 0-025 ml of glucose gelatin veronal buffer containing 1-5 x 10-4M Ca++ and 5 x 10' MMg++, pH 7*4 ionic strength 0 075; and six doubling dilutions were made. One drop of an appropriate dilution of complement from NHS was added and the mixture was incubated at 30°C for 30 min. One drop of EAC (1 x 108 sensitized sheep red blood cells/ml) and two drops of buffer were added and the mixture was incubated at 37°C in a shaker for 1 hr. The plates were then centri-
IgG complexes in primary biliary cirrhosis
fuged at 500 g for 10 min in a Model UV International centrifuge and examined visually for haemolysis. The presence of complexes in the fractions was determined by the Raji cell radioimmunoassay. Markers were IgG and IgM in serum determined by radial immunodiffusion (Mancini et al., 1965) with antisera made by us. Quantification of hepatic copper. The hepatic copper content was determined by atomic absorption spectroscopy (Perkin Elmer Corporation, 1973). Hepatitis-associated antigen. These determinations were made by the method of Ling & Overby (1972).
RESULTS Serum immune complexes by the Raji cell radioimmunoassay The Raji cell radioimmunoassay detects antigen-antibody complexes in serum by virtue of the binding of these complexes via receptors for complement on the surface of Raji cells (Theofilopoulos et al., 1976; Gupta et al., 1978). In our laboratory, sera of 100 normal healthy adults have been tested by this method and the upper normal limit has been found to be 55 gg AHG/ml. The levels of serum complexes determined on the initial examination of forty patients with PBC are presented in Fig. 1. Seventeen patients had elevated levels of complexes in their sera, and thirteen of these showed persistence of complexes over the two-year period (Fig. 2a). The sera of twenty-three patients with PBC had normal levels of complexes at the initial visit, but seven of these had elevated levels at 1 or 2 years, or both (Fig. 2b). In the three patients with initially elevated amounts whose levels dropped at 1 year to the upper limit of the normal range, elevated levels of complexes were detected once more at 2 years. Therefore, a total of twenty-four (60%) out of forty patients who had PBC had abnormal amounts of circulating complexes at some time during the study. The patients were therefore grouped as follows: group (a) twenty-four patients with elevated levels of complexes initially or during the follow-up; and group (b), sixteen patients with normal amounts of complexes both initially and subsequently. Since IgG may aggregate readily in hyperglobulinaemic stored sera and may result in falsely elevated levels of complexes by the Raji assay, we examined fresh sera from fifteen patients with PBC. We confirmed that levels of complexes are indeed elevated in PBC.
Histopathology ofthe liver and circulating complexes Table 1 shows a comparison of the amount of inflammatory cell infiltrate in the portal areas of liver biopsies in patients with persistently normal levels of complexes, group (b), and in patients with per-