Detection of antibodies to Chang liver cell in sera from patients with chronic liver diseases by 125I-labelled protein A binding assay and the effect of prednisolone and 6-mercaptopurine treatment on the level of the antibodies

Antibody binding to living Chang liver cell was measured in sera from 71 patients with various chronic liver diseases using 125I-labelled protein A binding assay. The level of antibody binding to Chang liver cell was significantly elevated in sera from patients with chronic active hepatitis (CAH), chronic persistent hepatitis (CPH) and liver cirrhosis as compared to those from healthy donors, but not in sera from patients with fatty liver. There was no detectable antibody binding to HeLa cells in those sera. The antibody binding to Chang liver cell was blocked by a human liver specific protein (LSP) preparation. The levels of antibody binding to Chang liver cell were significantly higher in patients with CAH than patients with CPH. On the other hand, the level of antibody binding to Chang liver cell was significantly decreased in sera from patients with CAH after a treatment with prednisolone (PSL) for 2 months and a subsequent combined administration of 6MP and a maintenance dose of PSL for 1 month. These results suggest that antibodies to Chang liver cell are closely correlated with the activity of chronic liver disease and that PSL and 6MP treatment can reduce the level of the antibodies.     

LM-Ag and LSP--two different target antigens involved in the immunopathogenesis of chronic active hepatitis?

Two different types of immune reactions against liver membrane antigens have been described: firstly, cell-mediated immunity and autoantibodies against the liver-specific protein (LSP) in HBsAg-negative and HBsAg-positive chronic active hepatitis (CAH), and secondly, a liver membrane autoantibody (LMA) in HBsAg-negative CAH. Using 100,000 g supernatants of human and rabbit liver homogenates, the corresponding antigen of LMA could be separated by affinity chromatography on insolubilized LMA-positive sera from patients with HBsAg-negative CAH. A further characterization by crossed immunoelectrophoresis showed that LMA is directed against a soluble liver membrane antigen (LM-Ag) that is not a constituent of the purified LSP. LM-Ag seems to be species-unspecific and moves faster than LSP and serum albumin in crossed immunoelectrophoresis. While LSP is believed to be involved in the pathogenesis of HBsAg-negative and HBsAg-positive chronic active hepatitis, LM-Ag may be a target antigen only in cases of HBsAg-negative autoimmune CAH.     

Identification of the hepatic asialo-glycoprotein receptor (hepatic lectin) as a component of liver specific membrane lipoprotein (LSP).

Liver specific membrane lipoprotein (LSP), the target for anti-LSP antibodies in various liver diseases, is thought to be comprised of fragments of the hepatocellular plasma membrane. In the present study, therefore, evidence has been sought for the presence in LSP of the hepatocyte surface receptor (hepatic lectin) that binds desialylated glycoproteins. Eight guinea-pig anti-LSP antisera (four anti-human and four anti-rabbit LSP) were found to react by ELISA and/or RIA against affinity purified human and rabbit hepatic lectin. Binding of the antisera to 125I-hepatic lectins was inhibited by the unlabelled lectins, by human and rabbit LSP and by purified rabbit liver plasma membranes but not by a 50,000-fold excess of kidney homogenate. The results indicate that hepatic lectin is a liver specific, species cross-reactive antigen comprising about 0.25% of the protein in LSP.     

Production of murine monoclonal antibodies against a liver-specific, species cross-reactive antigen in the liver-specific lipoprotein (LSP) preparation

Previous attempts in several laboratories have failed to produce murine monoclonal antibodies (MAbs) against liver-specific, species-cross-reactive, cell surface-expressed antigens in the normal liver preparation known as liver-specific membrane lipoprotein (LSP). In the present study, BALB/c mice were pretreated with a single dose of cyclophosphamide (20 mg/kg), hyperimmunized with human LSP and hybridomas produced by fusion of spleen cells from these mice with murine myeloma (P3-NS1-Ag4-1) cells. To bias selection in favour of MAbs reacting with species cross-reactive epitopes, hybridoma supernatants were screened by ELISA against rabbit LSP. From 70 stable hybridomas, four MAbs were obtained that react with rabbit LSP. One is an IgM antibody and the other three are of IgG2a class. All four react with the hepatic asialo-glycoprotein receptor (HL), a liver-specific, species-cross-reactive component that is normally expressed on the surfaces of hepatocytes. Using a rapid screening technique (an 'additive' ELISA), preliminary evidence was obtained indicating that these four MAbs between them recognise three different epitopes on the HL molecule. The results suggest that this is a viable approach for the production of MAbs against autoantigens to which autoreactivity may normally be suppressed.     

Study of cellular immunity in experimental autoimmune hepatitis in mice.

This study was undertaken to produce experimental autoimmune hepatitis in mice, and to examine the role of liver specific lipoprotein (LSP), if any, and of cellular immunity in such a model. After immunization of three strains of mice (C57BL/6, C3H/He and BALB/c) with syngeneic crude liver proteins, most prominent liver changes histologically mimicking human hepatitis were produced in the liver of C57BL/6 (B6) mice. Antigenic and immunogenic activity of LSP in the crude liver proteins was decreased by the treatment of freezing and thawing, and the recovery of the antigenic activity seemed to correlate with the susceptibility of immunized mice to the induction of liver damage. Autoantibody against LSP was demonstrated in the serum of immunized B6 mice, but not in the sera of other strains after immunization. It was also found that EDTA contained in the buffer used for purification of LSP distinctly suppressed lymphocyte activity in vivo and in vitro. With the use of EDTA free LSP, it was shown that spleen cells of immunized B6 mice (especially of T cell enriched fraction) had a high reactivity studied by lymphocyte transformation test. Further examination showed that EDTA free LSP could induce mild liver lesions and lymphocyte reactivity against LSP, although neither histological change nor lymphocyte reactivity was found in the liver of B6 mice immunized with EDTA containing LSP.     

中成药甘舒胶囊对抗氧化应激诱导的肝细胞损伤

目的探讨中药甘舒胶囊对抗氧化应激损伤的肝细胞保护作用。方法在Chang肝细胞建立氧化应激(H2O2)损伤的实验模型,应用甲氮甲唑蓝(MTT)检测法。PI染色流式细胞仪(FCM)及Hoechst 33258染色法等检测甘舒对抗H2O2诱导Chang肝细胞的细胞毒性及凋亡的细胞保护作用。结果H2O2呈浓度依赖性地降低Chang肝细胞的存活率;在自身不影响Chang肝细胞存活率的浓度(1～100μg/ml)范围内,甘舒呈浓度依赖性地对抗300μmol/L和400μmol/LH2O2对肝细胞存活率的抑制作用;另方面,在0～800μmol/L浓度范围内,H2O2呈浓度依赖性地增加Chang肝细胞的凋亡率;100μg/ml、500μg/ml和1mg/ml的甘舒本身不影响肝细胞的凋亡率,但却能显著地抑制300μmol/LH2O2诱导的肝细胞凋亡。结论中药甘舒胶囊具有抗氧化应激作用,可显著对抗氧化应激(H2O2)诱导的肝细胞损伤。     

Autoantibody responses to liver-specific lipoprotein in mice.

Autoantibody to the hepatocyte membrane antigen, liver-specific lipoprotein (LSP) was induced in mice by immunization with LSP-containing protein preparations from human, rat, rabbit and mouse liver and also with purified allogeneic LSP. Each of the strains of mice used (C57B1, BALB/c, C3H) showed the capacity to produce high titre autoantibody to LSP. Autoantibody to LSP demonstrated by passive haemagglutination was absorbed by normal mouse hepatocytes but not by kidney or spleen cells and reacted with the cell membrane of normal mouse hepatocytes by immunofluorescence. The liver was examined histologically in all mice and where inflammation was found it was attributable to the Freund's complete adjuvant used in immunization rather than liver protein immunogen. The demonstration of high titre autoantibody to LSP in mice without associated hepatitis contrasts with chronic hepatitis in man and experimental chronic hepatitis in rabbits where autoantibodies to LSP have been implicated in the pathogenesis of the disease.     

A human serum immunoglobulin with specificity for certain homologous target cells, which induces target cell damage by normal human lymphocytes

A factor has been found in a number of human sera which renders a polyploid strain of human liver cells, Chang cells, susceptible to damage by non-immune human lymphocytes. Sera possessing this factor are referred to as Factor Containing Sera (FCS). Such damage is assessed quantitatively by release of radioactive chromium from target cells. This factor has the chemical properties of IgG and can be absorbed out on Chang cells. Its specificity has been shown to be for Chang cells and not for human lymphocytes. Other homologous and heterologous target cells tested were not affected by this factor. The factor has not been shown to have any effect on Chang cell viability by itself, even in the presence of complement. Factors which inhibit target cell damage are shown to coexist with the factor which induces non-immune lymphocyte damage of Chang cells. The possible origin of this factor is discussed as is the role in immune reactions of target cell specific antibody which renders such cells susceptible to damage by non-immune lymphocytes.     

Detection and characterization of liver membrane autoantibodies in chronic active hepatitis by a solid-phase radioimmunoassay.

A solid-phase radioimmunoassay was developed to detect antibodies to liver membrane antigens in sera of patients with HBsAg-negative and -positive liver diseases and primary non-hepatic autoimmune diseases. Ten of fourteen patients with HBsAg-negative CAH had autoantibodies detected by RIA; negative results were obtained with sera of seven patients with HBsAg-positive acute and chronic liver diseases, six patients with miscellaneous liver diseases, including two patients with PBC, two healthy blood donors and seven patients with primary non-hepatic autoimmune diseases. Antibodies detected by RIA correlated with liver membrane autoantibodies (LMA) found by indirect immunofluorescence; no correlation was observed with AMA, ANA and SMA. Species-cross-reacting antibodies could be absorbed by preincubation with isolated plasma cell membranes prepared from rabbit livers. Liver membrane autoantibodies detected by RIA were directed against three different antigen fractions obtained from Sepharose 6B chromatography including LSP and LM-Ag. Only three of ten antibodies were directed against species-specific determinants; others cross-reacted with rabbit antigens. Only the antibody to LSP was organ-specific, all others cross-reacted with kidney proteins. Ferritin, human serum albumin and human plasma lipoprotein were excluded as target antigens. Although several sera reacted with identical molecules a remarkable heterogeneity of liver membrane autoantibodies was observed.     

Incidence of antibodies against rabbit liver specific lipoprotein (RLSP) in chronic active hepatitis.

In chronic active hepatitis (CAH) evidence exists that circulating autoantibodies against liver specific lipoprotein (LSP) could play a role in the development of hepatocellular injury. We evaluated the presence of autoantibodies in CAH against LSP using rabbit LSP, as antigen in a radioimmunoprecipitation test. Fifty-one patients with histologically diagnosed CAH were investigated. Among these 16 were HBsAg+, 15 were HBsAg-/anti-HBc+, 10 were non-A, non-B, 10 were autoimmune CAH. Anti-LSP were detected in six of 16 (37%) HBsAg+ (mean titre of 1:198); four of 15 (33%) HBsAg-/anti-HBc+ (mean titre of 1:246); two of 10 (20%) non-A, non-B (mean titre of 1:185); seven of 10 (70%) autoimmune CAH (mean titre of 1:307). No correlation was evident between the titre of anti-LSP and the values of AST, bilirubin or IgG. The findings seem to be consistent with the following conclusions: (a) CAH patients develop an humoral immune response to determinants in LSP which are not species specific. This is further evidence that rabbit LSP could be considered a suitable alternative to the human preparation in evaluation of autoimmunity in CAH and (b) the different behaviour of anti-LSP in patients with viral CAH (B, non-A, non-B) in respect of patients with autoimmune CAH suggests a variable importance of these antibodies in the mechanism of ongoing liver cell injury according to the various types of CAH.     

Cytotoxic Effect of an Anti-Liver Monoclonal Autoantibody obtained after Neonatal Thymectomy in Mice

A monoclonal autoantibody, LSA-1, against murine liver antigen was obtained by fusing spleen cells from a neonatally thymectomized BALB/c mouse with SP2/0 murine myeloma cells. The LSA-1 isotype was IgG2b and kappa. LSA-1 was specific to the liver, especially, to a liver-specific membrane lipoprotein (LSP) fraction. By Western blotting analysis, LSA-1 mainly detected a 100 kDa protein of LSP fraction. LSA-1 stained cytoplasm of the cryostat sections of liver in immunohistochemical analysis. Furthermore, the antigen recognized with LSA-1 was highly expressed on the surface of a murine hepatoma cell line, MH134, slightly on a murine normal liver cell line, C1469, and on freshly prepared hepatocytes, but not on spleen cells. LSA-1 had a cytotoxic activity on liver cell lines in the presence of a complement in vitro. Furthermore, injection of LSA-1 into mice-induced liver injury. These results suggest that anti-liver autoantibody plays an important role in the induction of autoimmune hepatitis. Accordingly, this antibody will be a useful tool for the analysis of the pathogenesis of autoimmune hepatitis.     

ADCC against human erythrocyte target cells: role of the anti-target cell antibodies in determining lymphocyte killer activity.

Studies were undertaken to investigate the role of anti-target cell antibodies in determining whether lymphocytes can mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro. Trinitrophenyl (TNP) modified Chang liver cells and human erythrocytes were employed as target cells and were coated with xenogeneic and allogeneic antibodies against TNP and natural cell surface antigens. Two cytotoxic effector cell populations were used: human peripheral blood mononuclear cells (PBMC) containing both lymphocytes and monocytes, and monocyte-depleted peripheral blood lymphocytes (PBL). With Chang targets, both PBMC and PBL mediated ADCC with xenogeneic anti-Chang and xenogeneic anti-TNP sera. With human erythrocyte targets, PBMC but not PBL mediated ADCC with human anti-blood group B serum, while both PBMC and PBL mediated ADCC with xenogeneic anti-TNP sera and also with a human anti-CD serum. These results demonstrate that the source of anti-target cell antibodies employed in ADCC reactions may determine whether or not lymphocytes are capable of mediating cytotoxicity.     

Studies on experimental chronic active hepatitis in the rabbit. II. Immunological findings.

The role played by humoral and cellular immune response to liver antigens in the pathogenesis of experimental chronic active hepatitis (CAH) was studied in rabbits which had been immunized with the two human liver-specific proteins LSP and LP2, with LSP alone, or with an extract of human skeletal muscle. Rabbits immunized with LSP, alone or with LP2, developed skin test reactivity and circulating antibody to homologous LSP; liver biopsy revealed immunoglobulin bound to the hepatocyte cell surface. It has been suggested that cellular immunity to homologous LSP or alternatively, antibody to an antigenic determinant shared by human and rabbit LSP, may play a role in the pathogenesis of experimental CAH, but both abnormalities were present in two rabbits which did not develop CAH despite observation for at least 18 months. Five normal rabbits given an intravenous injection of serum pooled from rabbits with CAH did not develop significant hepatic lesions. Immunity to homologous LSP or other hepatocyte cell surface antigens could not be detected in any rabbit which had been immunized with skeletal muscle, and hepatocytes from these rabbits did not have immunoglobulin on their cell surface. The pathogenesis of CAH in these animals is obscure. These findings suggest that mechanisms other than an auto-immune response to LSP play an important role in the pathogenesis of experimental chronic active hepatitis.     

Studies on experimental chronic active hepatitis in the rabbit. II. Immunological findings

The role played by humoral and cellular immune response to liver antigens in the pathogenesis of experimental chronic active hepatitis (CAH) was studied in rabbits which had been immunized with the two human liver-specific proteins LSP and LP2, with LSP alone, or with an extract of human skeletal muscle. Rabbits immunized with LSP, alone or with LP2, developed skin test reactivity and circulating antibody to homologous LSP; liver biopsy revealed immunoglobulin bound to the hepatocyte cell surface. It has been suggested that cellular immunity to homologous LSP or alternatively, antibody to an antigenic determinant shared by human and rabbit LSP, may play a role in the pathogenesis of experimental CAH, but both abnormalities were present in two rabbits which did not develop CAH despite observation for at least 18 months. Five normal rabbits given an intravenous injection of serum pooled from rabbits with CAH did not develop significant hepatic lesions. Immunity to homologous LSP or other hepatocyte cell surface antigens could not be detected in any rabbit which had been immunized with skeletal muscle, and hepatocytes from these rabbits did not have immunoglobulin on their cell surface. The pathogenesis of CAH in these animals is obscure. These findings suggest that mechanisms other than an auto-immune response to LSP play an important role in the pathogenesis of experimental chronic active hepatitis.     

Lymphocyte-specific protein 1: a specific marker of human leucocytes

While both murine and human homologues of the LSP1 gene (lymphocyte-specific gene 1) and its protein products have been identified, studies on human LSP1 have been limited. The present report describes a detailed immunocytochemical study of the distribution and localization of human LSP1 in both normal and neoplastic cells and tissues. The specificity of the monoclonal anti-LSP1 reagent was confirmed by expression cloning and transfection studies. The intracellular 60 000 MW LSP1 protein was found to be present in peripheral blood B cells, monocytes and granulocytes but absent in a subpopulation of circulating T cells (10-15% of CD3-positive T cells). The presence of LSP1 protein in medullary thymocytes, but only in scattered cortical thymocytes, provided additional evidence for heterogeneity of expression in T cells. Novel observations also included the presence of LSP1 in plasma cells, dendritic cells and Langerhans' cells. The leucocyte-restricted distribution of LSP1 protein means that it may play an important role in haematopathology. LSP1 protein was detected in a wide range of leukaemias and lymphomas, particularly of B-cell origin, and in tumour cells in classical Hodgkin's disease. Of interest was the indication of a reciprocal relationship in the expression of LSP1 and ALK (anaplastic lymphoma kinase) proteins in patients with anaplastic large cell lymphoma. As the anti-LSP1 reagent used in the present study recognizes a formalin-resistant epitope it should be of considerable value in the diagnosis of routinely fixed material.     

Human antibody-dependent cellular cytotoxicity and natural killer cytotoxicity to herpes simplex virus-infected autologous and allogeneic cells.

Using cultured skin shavings, human cellular cytotoxicity to uninfected and herpes simplex virus (HSV)-infected autologous and allogeneic fibroblasts and Chang liver cells was analysed in a 51Cr release assay. The effector cell requirements and characterization, time kinetics and antibody requirements were similar using each HSV-infected target cell in an antibody-dependent cellular cytotoxicity (ADCC) system. There was lower natural killer cytotoxicity (NKC) to uninfected autologous cells than unrelated cells in an 18 hr assay. NKC to infected autologous and unrelated fibroblasts was similar to that mediated against Chang liver cells. Thus NKC to uninfected fibroblasts correlated with the relationship of effector and target cells while NKC to infected cells correlated with the intrinsic lytic potential of the effector cells. The autologous system offers little advantage in the analysis of ADCC or NKC in normal individuals to virus-infected cells, but is probably crucial for the detection of HLA-restricted T-cell cytotoxicity. The demonstration of autologous anti-viral ADCC and NKC lends further credence to the in vivo importance of the mechanisms.     

Effect of neonatal thymectomy on experimental autoimmune hepatitis in mice.

Using an autoimmune hepatitis model of A/J mice which was prepared with immunization by syngeneic crude liver proteins, various influences of neonatal thymectomy were studied by observations of histological liver changes, autoantibody to liver-specific membrane lipoprotein (LSP), delayed-type hypersensitivity (DTH) to LSP, and purified protein derivative (PPD), and suppressor activity to LSP. The liver changes in the thymectomized mice were more intense than those in the non-thymectomized controls. Production of the anti-LSP autoantibodies and positive DTH to syngeneic LSP could be recognized in both groups of the thymectomized mice and the non-thymectomized controls, but the levels of those were higher in the former. In the level of DTH to PPD the thymectomized mice were lower than the non-thymectomized controls. Adoptive transfer experiments showed that suppressor activity to LSP was reduced in the spleen cells of neonatally thymectomized mice. This experiment suggests that neonatal thymectomy is apt to abolish tolerance to LSP on account of depressed suppressor activity to autoantigen, and accordingly liver damage is increased.     

Differential thioredoxin reductase activity from human normal hepatic and hepatoma cell lines

Thioredoxin reductase (TrxR), a component of the thioredoxin system, including thioredoxin (Trx) and NADPH, catalyzes the transfer of electrons from NADPH to Trx, acts as a reductant of disulfide-containing proteins and participates in the defense system against oxidative stresses. In this study, the regulation pattern of TrxR in the presence of various stressful reagents was compared between Chang (human normal hepatic cell) and HepG2 (human hepatoma cell) cell lines. Aluminum chloride (0.5 mM) and zinc chloride (0.5 mM) enhanced the TrxR activity in the Chang cell line to a higher degree than in the HepG2 cell line, but cupric chloride (0.2 mM) and cadmium chloride (0.1 mM) enhanced the TrxR activity in the HepG2 cell line to a greater degree. The TrxR activities in both Chang and HepG2 cell lines were similarly induced by treatment with sodium selenite (0.02 mM) and menadione (0.5 and 1.0 mM). Lipopolysaccharide (2 micro g/m1) increased the TrxR activity upto 4.02- and 2.2-fold in the Chang and HepG2 cell lines, respectively, in time-dependent manners. Hydrogen peroxide (5 mM) markedly enhanced the TrxR activity in the HepG2 cell line, but not in the Chang cell line. NO-generating sodium nitroprusside (3.0 and 6.0 mM) induced TrxR activities in both human liver cell lines. The TrxR activity was also induced in human liver cells under limited growth conditions by serum deprivation. These results imply that the TrxR activities in normal hepatic and hepatoma cell lines are subject to different regulatory responses to various stresses.     

Induction of autoimmune hepatitis and autoantibodies to liver antigens by neonatal thymectomy in mice

We examined development of autoimmune hepatitis in neonatally thymectomized C3H/HeN mice and tried to characterize the nature of liver antigens recognized by the autoantibodies at the molecular level. Autoantibodies to crude liver proteins detected by ELISA were found in 12 (67%) of 18 mice thymectomized 2 days after birth. However, autoantibodies were not detected in mice thymectomized 7 days after birth. The autoantibodies mainly consisted of IgG and reached the maximum level 8 weeks after birth. Hepatic inflammation, mononuclear cell infiltration in the portal area, was seen in 5 (28%) of 18 mice thymectomized 2 days after birth, but not in mice thymectomized 7 days after birth. Most infiltrating cells were Thy-1⁺ lymphocytes. The serum autoantibody level to crude liver proteins in mice with hepatitis was much higher than that in mice without hepatitis. We fractionated crude liver proteins by a Sepharose 6B column and examined the reactivity against the autoantibodies. The autoantibodies of three of five mice with hepatitis reacted with the ≈150 kD liver proteins other than liver-specific protein (LSP). By Western immunoblotting of SDS–PAGE using LSP and fractionated liver proteins, we found that the molecular weights of the target antigens were 52 kD in LSP and 150 kD (strong band), 138, 128, 120 and 110 kD (weak band) in fractionated liver proteins other than LSP. This 150-kD target molecule in crude liver proteins was found only in liver. These results indicate that hepatitis and autoantibodies to liver proteins are induced spontaneously by neonatal thymectomy in mice, and the candidates of autoantigen in this hepatitis model are 52-kD protein in LSP and 150-kD liver proteins different from LSP. Still more, we regard the 150-kD molecule as a new autoantigen related to hepatitis.     

Immunohistochemical localization of human liver specific protein using rabbit antisera and the avidin-biotin complex technique.

A sensitive enzymatic avidin-biotin complex technique was utilized to locate the antigenic sites in human liver which react with rabbit antibodies to human liver specific protein (LSP). Depending on the rabbit antiserum used, anti-LSP reactive material was seen in different locations. (1) diffusely distributed in liver parenchymal cells, (2) in both hepatocyte cytoplasm and plasma membrane and (3) in hepatocyte plasma membrane only. The cytoplasmic staining was partially or totally abolished by preabsorption with human kidney homogenate whilst the plasma membrane staining remained uninfluenced. The latter was apparently confined to bile canalicular walls and may be liver specific in contrast to the cytoplasmic reaction which seems to be due to antigen determinants cross-reacting with tissue components in kidney.