Differences in immune recognition of cytochrome P4502D6 by liver kidney microsomal (LKM) antibody in autoimmune hepatitis and chronic hepatitis C virus infection.

LKM-1 antibody, which characterizes a subtype of autoimmune hepatitis (AIH), is also found in some patients with chronic hepatitis C virus (HCV) infection. It has been suggested that HCV initiates autoimmunity through molecular mimicry, because there is partial identity between HCV and cytochrome P4502D6 (CYP2D6), the putative target of LKM-1. Whether CYP2D6 is the target of LKM-1 in HCV-related liver disease, however, is controversial. To clarify this issue, we have studied by phage plaque assay and Western blot the reactivity to recombinant CYP2D6, isolated from a human liver cDNA library, in 55 patients with LKM-1, 18 (14 females, median age 12 years) anti-HCV-negative, with classical AIH, and 37 (27 females, median age 52 years) anti-HCV-positive. Reactivity to CYP2D6 was found in 72% of the anti-HCV-negative, but only in 27% of the anti-HCV-positive patients (P < 0.001), although immunofluorescence LKM-1 titres were similar in the two groups. In addition, to investigate whether the antibody responsible for the LKM-1 fluorescent pattern also reacts with CYP2D6, we have determined the specificity of LKM-1 antibodies present in the supernatant of lymphoblastoid B cell lines obtained from two patients with LKM-1-positive AIH. An oligo/monoclonal antibody thus generated gave both the typical fluorescent pattern and reacted with CYP2D6. Our results show that whilst antibodies producing the characteristic LKM-1 fluorescent pattern can react with CYP2D6, not all LKM-1-positive sera do so, particularly if obtained from patients with chronic HCV infection. This suggests that LKM-1 in HCV infection recognizes epitopes or antigens different from those targeted in AIH.

Superantigens as immunogens and tolerogens.

Superantigens are a family of proteins that are receiving a great deal of attention as a group of potent immunomodulatory molecules. They combine with MHC class II molecules to form the ligands that stimulate T cells via the V beta element of the T cell receptor. Two groups of superantigens have been described: those expressed endogenously such as the mouse mammary tumour virus (MMTV) and the exogenously derived products such as the Staphylococcal enterotoxins. Here we review the interactions between the superantigens and MHC class II molecules, the mechanisms of T cell activation, the role of superantigens in the induction of tolerance and their involvement in autoimmune diseases.

T lymphocyte activation is associated with viral replication in chronic hepatitis B virus infection of childhood.

Activated circulating T lymphocytes expressing HLA-DR (mean +/- s.d. 11.0 +/- 5.2%) or interleukin-2 receptor (IL-2R) (2.1 +/- 1.7%) were significantly increased in 63 children with chronic hepatitis B virus (HBV) infection when compared with 33 age-matched healthy controls (3.0 +/- 1.3%, P less than 0.01, and 0.1 +/- 0.4%, P less than 0.01). HBeAg-positive patients had higher percentage of DR (11.9 +/- 5.1%) or IL-2R (2.4 +/- 1.7%) positive T lymphocytes than anti-HBe-positive children (7.4 +/- 3.6% and 1.1 +/- 1.5%, P less than 0.01 and P = 0.02 respectively). Similarly, HBV DNA-positive patients had higher percentage of DR (10.5 +/- 3.3) or IL-2R (3.2 +/- 1.7%) positive T cells than HBV DNA-negative children (6.6 +/- 2.8% and 1.2 +/- 1.5%, P less than 0.01 for both). There was a positive correlation between percentage of DR positive T lymphocytes and levels of HBV DNA. Sixty-two per cent of the DR-positive T lymphocytes were cytotoxic/suppressor and 35% helper/inducer. The relationship between viral replication and T lymphocyte activation is discussed.

Autoimmune chronic active hepatitis in a family.

A positive family history of autoimmune disease is common among patients with autoimmune chronic active hepatitis, but usually autoimmunity is directed at organs other than the liver. We document for the first time the multiple occurrence of autoimmune chronic active hepatitis in a family. Out of a sibship of seven, three sisters developed this, one sister developed coeliac and autoimmune thyroid disease, one sister showed serological signs of autoimmunity, while the two brothers were well with no signs of autoimmunity. HLA typing showed that in association with the female sex DR3 seems to be more important than B8 in conferring susceptibility to autoimmune chronic active hepatitis, at least in this family.

Class I and class II major histocompatibility complex antigens on hepatocytes: importance of the method of detection and expression in histologically normal and diseased livers.

Methodological differences in major histocompatibility complex (MHC) antigen detection were investigated on isolated, viable hepatocytes and cryostat hepatic sections from 27 children with liver disorders, six of whom had normal histology. Class I antigens were constantly found on sections using a three step immunoperoxidase technique after acetone/chloroform fixation, other techniques being less sensitive, or on isolated hepatocytes by indirect immunofluorescence alone. With mechanical isolation the percentage of positivity ranged from 85 to 100%, while with collagenase isolation it ranged from 22 to 49% on immediate testing, and from 53 to 80% after 24 hour incubation. Class II antigens were only detected in one patient with autoimmune chronic active hepatitis and two with primary sclerosing cholangitis. Flow cytofluorimetric analysis in 11 cases confirmed class II or class I positivity, or both, on isolated hepatocytes, allowing MHC antigen expression on hepatocytes to be measured. Class I and II antigen detection on hepatocytes is influenced by the technique used. Although class I antigens are invariably expressed on hepatocytes, class II antigens are only found on hepatocytes from patients with immune mediated liver disorders.

T-cell-directed hepatocyte damage in autoimmune chronic active hepatitis.

To investigate the function of activated T lymphocytes in autoimmune chronic active hepatitis, 7 of 15 T-cell clones from the peripheral blood of 8 patients were studied. These clones showed specificity for liver-membrane antigen with proliferation when stimulated by rabbit liver cell membranes. 6 of these clones reacted with liver-specific lipoprotein complex, and 1 clone (and 3 subclones) responded to the asialoglycoprotein receptor (ASGPR), both known targets of immune attack in autoimmune chronic active hepatitis. 2 of these clones stimulated autologous B lymphocytes to produce liver-membrane-specific autoantibodies and antibody to the ASGPR. These results suggest that liver-membrane-specific activated T lymphocytes in peripheral blood may be important in the autoimmune attack of chronic active hepatitis.

Class I and class II major histocompatibility complex antigen expression on hepatocytes: a study in children with liver disease.

Controversy exists regarding major histocompatibility complex antigen expression on hepatocytes. In this study, hepatocyte expression of class I and II major histocompatibility complex antigens was investigated in diseased and normal livers, using indirect immunofluorescent staining of mechanically isolated, viable hepatocytes. Hepatocytes were obtained from 76 children: 10 with autoimmune chronic active hepatitis, nine with primary sclerosing cholangitis, nine with chronic hepatitis B virus infection, five after liver transplantation, 19 with extrahepatic biliary atresia, 11 with alpha 1-antitrypsin deficiency, four with idiopathic neonatal hepatitis and nine with histologically normal liver. Immunohistochemistry was performed in all cases; flow cytofluorimetry was performed for class I antigens in 38 cases and performed for class II antigens in 18 cases. From three children with autoimmune chronic active hepatitis and two with chronic hepatitis B virus infection, isolated hepatocytes were also incubated with gamma-interferon before staining and analysis. By fluorescence microscopy, class I antigens were detected on hepatocytes from all children, the highest percentage (100%) of positive cells and the most intense staining were observed in untreated patients with autoimmune chronic active hepatitis or primary sclerosing cholangitis and in those with acute rejection of a liver transplant. Reduced class I antigen expression occurred in chronic hepatitis B virus infection. Class II antigens were only detected on hepatocytes from eight patients: three with autoimmune chronic active hepatitis and five with primary sclerosing cholangitis, all untreated. Flow cytofluorimetric analysis confirmed the results obtained by fluorescence microscopy, but it also demonstrated a weak class II antigen expression during liver allograft rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble interleukin 2 receptors in autoimmune chronic active hepatitis.

Children with uncontrolled autoimmune chronic active hepatitis have increased numbers of activated T lymphocytes expressing interleukin 2 receptors (IL2R). A soluble form of IL2R has recently been described whose proposed role is to downregulate T cell activation by competing for interleukin 2. We investigated whether a deficiency of soluble IL2R could account for the high concentrations of IL2R positive T lymphocytes in autoimmune chronic active hepatitis. Soluble IL2R was measured by enzyme-linked immunosorbent assay in the serum of 16 children with autoimmune chronic active hepatitis, eight with chronic liver disease due to hepatitis B virus infection, seven with Wilson's disease, nine with alpha 1 antitrypsin deficiency, and 15 healthy age matched controls. Soluble IL2R concentration was significantly higher in patients with autoimmune chronic active hepatitis than in healthy controls (mean (SEM) 475 (75) U/ml, 145 (8) U/ml respectively, p less than 0.01). Eleven patients who had active disease had significantly higher soluble IL2R concentrations (590 (89) U/ml) than the five cases with inactive disease (220 (36) U/ml, p less than 0.01). No difference was found between the controls and the patients with chronic liver disease due to hepatitis B infection, Wilson's disease, and alpha 1 antitrypsin deficiency. Percentages and absolute numbers of surface IL2R positive T cells as detected by immunofluorescence were significantly higher in the patients with autoimmune chronic active hepatitis (11.8% (1); 274/microliters (31)) than in controls (0.2% (0.1); 5/microliters (2), p less than 0.001), the highest values being found in those with uncontrolled disease. A significantly positive correlation was observed between concentrations of soluble IL2R and the percentage of T cells expressing IL2 receptors (r=0.67, p<0.001). These results indicate that the high levels of IL2R positive T lymphocytes characteristic of autoimmune chronic active hepatitis are not due to a deficiency of soluble IL2 receptors.

Different immune mechanisms leading to autoimmunity in primary sclerosing cholangitis and autoimmune chronic active hepatitis of childhood.

Children with primary sclerosing cholangitis or autoimmune chronic active hepatitis have similar high levels of immunoglobulin G and non-organ-specific autoantibodies and may have similar histological features. To investigate a possible immunopathogenesis of primary sclerosing cholangitis, we have studied a series of regulatory and/or effector immune mechanisms in eight children with primary sclerosing cholangitis, comparing them to 14 children with autoimmune chronic active hepatitis and 24 healthy children as controls. Antibodies to a liver membrane protein preparation were found in all children with autoimmune chronic active hepatitis tested and in seven of eight with primary sclerosing cholangitis, whereas antibodies against the hepatic asialoglycoprotein receptor were present in three of six patients with autoimmune chronic active hepatitis and in two of the eight with primary sclerosing cholangitis. Lymphocyte cytotoxicity values to autologous hepatocytes were similarly elevated in primary sclerosing cholangitis (median: 50%; range: 38 to 83%) and in autoimmune chronic active hepatitis (median: 52%; range 37 to 87%) compared to controls (median: 8%; range: 0 to 27%) (p less than 0.01 for both). In contrast, T suppressor cell number and function were normal in patients with primary sclerosing cholangitis (median: 23%; range: 19 to 28%; and median: 54%; range: 44 to 61%), but significantly decreased in patients with autoimmune chronic active hepatitis (median: 15%; range: 9 to 21%; and median: 9%; range: -40 to +21%) when compared to controls (median: 24%; range: 16 to 29%; and median: 53%; range: 8 to 77%) (p less than 0.01 for both).(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of anti-liver cell membrane antibody using a human hepatocellular carcinoma cell line.

A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of 125I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

IgG subclass deficiency in asthma.

Total immunoglobulin G (IgG) and subclasses were measured in serum samples from 82 children with chronic asthma, aged 1.5 to 6.3 years, and 76 controls. Concentrations of IgG1, IgG2, IgG3, and total IgG were significantly lower in asthmatic children aged 1 to 5, and IgG2 concentrations were also significantly lower in asthmatic children over 5 years of age. Twenty eight asthmatic children had at least one value in the deficient range, and 26 had IgG2 deficiency alone or in combination. Five children had IgG2 and IgA deficiency. These 28 children were significantly younger and fewer had raised IgE concentrations than the remainder. IgG subclass deficiency, which may reflect delayed maturation of the immune system, is common in young asthmatic children, and may have a role in the pathogenesis of the disease.

Proteinuria and activated T-lymphocytes in diabetic nephropathy.

The reasons for the presence of activated T-lymphocytes (ATL) in some long-standing insulin-dependent diabetic (IDDM) patients are unknown. These cells have been implicated in the genesis of proteinuria in some forms of immune-mediated renal disease. We measured ATL in 18 IDDM patients with diabetic nephropathy, 10 with nonnephrotic proteinuria (total urinary protein excretion rate greater than 0.5 and less than 3.5 g/24 h) and 8 with nephrotic proteinuria (total urinary protein excretion rate greater than 3.5 g/24 h), and in 17 age-, sex-, and duration-of-diabetes-matched diabetic control subjects without clinical proteinuria (total urinary protein less than 0.5 g/24 h). T-lymphocytes purified from peripheral blood were stained by direct immunofluorescence with the fluorescein-labeled monoclonal antibody anti-HLA-DR. Absolute number and percent of DR-positive T-lymphocytes were significantly higher in patients with nonnephrotic proteinuria (median and range 162 x 10(6)/ml, 40-320 x 10(6)/ml; 13.9%, 8.1-19.4%) compared with nonproteinuric control subjects (81 x 10(6)/ml, 2-240 x 10(6)/ml, P less than .05; 6.2%, 0-13.1%, P less than .01). In 8 patients with nephrotic proteinuria, absolute and percent DR-positive T-lymphocytes tended to be lower (36 x 10(6)/ml, 14-56 x 10(6)/ml; 3.4%, 1.1-5.4%) than in nonproteinuric control subjects. An increased number of activated T-lymphocytes may be part of an immune-mediated process associated with the development of proteinuria in diabetic nephropathy. In advanced renal disease with nephrotic proteinuria, this immune process may become exhausted.

Evidence of impaired antigen non-specific but normal antigen specific suppressor cell function in children with autoimmune chronic active hepatitis.

In autoimmune chronic active hepatitis (aCAH), autoaggression is believed to derive from a defect in immunoregulation. Antigen non-specific Concanavalin A (Con A) induced suppressor cell function has been reported to be impaired. In 11 children with aCAH we have investigated inhibition of production of a specific antibody (anti-tetanus toxoid, anti-TT) by suppressor cells induced either by a non-specific stimulus (Con A) or by the specific antigen (tetanus toxoid, TT). Con A induced suppression of anti-TT was significantly lower in patients (15.7 +/- 2.5%) than in controls (46.7 +/- 4.4%; P less than 0.01). In contrast, high dose tetanus toxoid induced suppression was similar in patients and controls (69.8 +/- 4.2, 72.0 +/- 3.6%, respectively). Both groups had similar serum anti-TT levels and in vitro production of anti-TT in response to optimal dose of TT. Our data indicate that antibody production to a T cell-dependent antigen is under the control of at least two regulatory mechanisms, one antigen specific and one antigen non-specific, only the latter being defective in aCAH.

Quantification of C3d in biological fluids by an enzyme-linked immunosorbent assay.

Using a commercial source of peroxidase-labelled anti-C3d antibody (Dakopatts), an enzyme-linked immunosorbent assay (ELISA) has been developed to quantify the complement fragment C3d. The technique enables the detection of C3d in plasma, urine and cerebrospinal fluid (CSF). The C3d-ELISA therefore provides a very sensitive technique for the evaluation of complement activation in biological fluids. In both plasma and urine the technique is able to discriminate between samples from normal controls and patients with rheumatoid arthritis in whom complement activation is known to occur. A good correlation was found between results obtained by ELISA and those by laser nephelometry (r = 0.91, P less than 0.0001). Microtitre plates pre-coated with anti-C3d antibody and subsequently stored at -70 degrees C retained the ability to perform in this assay. The sensitivity, short assay time and use of commercial reagents and pre-coated plates give this technique numerous potential applications in the evaluation of complement activation.

Characterization of anti-liver kidney microsomal antibody in childhood autoimmune chronic active hepatitis: evidence for IgG1 subclass restriction, polyclonality and non cross-reactivity with hepatocyte surface antigens.

An indirect immunofluorescence technique was used to investigate the immunoglobulin class, IgG subclass, light chain type and complement fixing ability of anti-liver kidney microsomal antibody (anti-LKM) in the sera of six children and five adults with autoimmune chronic active hepatitis (aCAH). Anti-LKM titres ranged from 1/80 to 1/20,480. In the children, the antibody belonged to the IgG1 subclass alone (titre 1/80-1/20,480) and was able to fix complement (titre 1/40-1/5120). In the adult group, antibody belonged to the IgG1 subclass in three cases (titre 1/40-1/640) whilst two belonged to both IgG1 (titre 1/640) and IgG4 (titre 1/40, 1/640). Such subclass restriction is similar to that found in other autoimmune disorders and may be genetically determined. Investigation of the light chain constituent of anti-LKM revealed that the kappa to lambda ratio was consistent with a polyclonal antibody response. To investigate the nature of the antigen to which anti-LKM is directed, the ability of these sera to bind to the surface membrane of isolated human hepatoma cells (Alexander cells) was investigated. Four of the eleven sera showed significant binding activity. Prior incubation of these four sera with Alexander cells abolished their membrane binding activity, but did not alter the anti-LKM titre. These results suggest that anti-LKM binds to cytoplasmic constituents alone and does not cross-react with surface antigens.

Preferential activation of helper/inducer T lymphocytes in autoimmune chronic active hepatitis.

We found a significant increase of activated circulating T lymphocytes expressing interleukin 2 receptor (IL-2r) (mean +/- s.e.m. 11.0 +/- 1.1%) or DR antigen (5.0 +/- 0.49%) in patients with autoimmune chronic active hepatitis (CAH) starting in childhood when compared to healthy controls (0.14 +/- 0.09%, P less than 0.001 and 2.8 +/- 0.06%, P less than 0.01). Patients with liver disorders due to Wilson's disease (IL-2r 0.64 +/- 0.25%, DR 3.5 +/- 0.22%) or alpha-1-antitrypsin deficiency (IL-2r 0.1 +/- 0.06%, DR 2.8 +/- 0.35%) had levels similar to controls. Levels of both IL-2r and DR positive T lymphocytes were higher in patients with uncontrolled CAH (IL-2r 18.0 +/- 1.01%; DR 6.3 +/- 0.78%) than in patients with inactive disease (IL-2r 3.2 +/- 1.4%, P less than 0.001; DR 3.0 +/- 0.13%, P less than 0.01). In patients with active disease levels of IL-2r positive cells were higher than DR positive cells (P less than 0.001). Only 21% of activated T cells coexpressed the two markers of activation. Sixty-seven percent of IL-2r positive T lymphocytes were helper/inducer and 25% suppressor/cytotoxic, while 66% of the DR positive T cells were suppressor/cytotoxic and 31% helper/inducer. The finding that the highest levels of activated T lymphocytes are present in patients with uncontrolled CAH suggests that these cells are involved in its pathogenesis. The preferential increase of activated helper/inducer cells might explain the enhanced immune reactivity characteristic of autoimmune CAH.

T suppressor cell function and number in children with liver disease.

Con A stimulated suppressor cell function and the proportion of suppressor T cells were reduced in children with untreated chronic active hepatitis (CAH) but were normal in corticosteroid treated CAH patients, patients with severe acute hepatitis and inactive chronic liver disease. Adults with CAH also have defective suppressor function but a normal proportion of T suppressor cells. This difference may account for the observation that relapse after treatment withdrawal is less frequent in children than in adults.

Immunoregulation of immunoglobulin production in normal infants and children.

Proportion of T cell subsets, spontaneous and PWM stimulated immunoglobulin production by peripheral blood lymphocytes and concanavalin A- (Con A) stimulated suppressor cell activity on immunoglobulin production by B cells was studied in 37 infants and children, to investigate changes of these parameters with age. Proportion of suppressor/cytotoxic (T8+) T lymphocytes was significantly lower in children below the age of 5 years, while there was no difference in proportion of total T lymphocytes (T3+) and helper/inducer (T4+) T cells. Spontaneous production and secretion of IgG and IgM by lymphocytes from children of all age groups was similar to that found in adults, but lymphocytes of children below the age of 10 years showed a low response to PWM stimulation. The activity of Con A-induced suppressor cells in inhibiting B cells producing immunoglobulins was almost absent in infancy, gradually increased during childhood and reached adult levels in teenagers. A significant association between the proportion of T8+ cells and Con A-induced suppression of B cell proliferation and a relationship between T4+ cells and spontaneous Ig production indicated the increasing maturity with respect to both number and function of peripheral blood lymphocyte subsets with advancing age.