RESUMEN
Although Graves' disease (GD), Hashimoto's thyroiditis (HT) and idiopathic myxoedema (Myx) are clinically distinct autoimmune thyroid diseases, previous studies using crude thyroid preparations as a source of antigens have failed to identify differences in cellular immune responses. In this study, we have assessed cellular immunity in patients with these disorders to two antigen preparations (derived from thyroid gland and cervical fat) enriched in cell membranes and known to share a functional TSH receptor. Using an indirect T-lymphocyte migration inhibitory factor (T-LIF) assay, T-cell immunity to thyroid membranes was demonstrated in 11/11 patients with GD, 4/5 with HT and 4/5 with Myx, but in none of 18 patients with chronic active hepatitis (another organ-specific autoimmune disease) or sixteen healthy controls. In contrast, T lymphocytes responsive to adipocyte membranes were detected only in patients with GD. TSH binding inhibiting antibodies were found exclusively in six patients with GD, and thyroid stimulating antibodies in five of these patients. In co-culture experiments designed to study the activity of antigen-specific suppressor T cells, low numbers of T cells from 6/6 normal controls, 4/4 patients with HT and 5/5 patients with myxoedema suppressed the response to adipocyte membranes of T lymphocytes from patients with Graves' disease. The results of this study demonstrate different patterns of T-cell reactivity to thyroid antigens in patients with Graves' disease, Hashimoto's thyroiditis and myxoedema and suggest that cellular immunity to the TSH receptor is restricted to patients with Graves' disease and associated with a defect in the specific immunoregulatory control of this response.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedad de Graves/inmunología , Receptores de Tirotropina/inmunología , Linfocitos T/inmunología , Glándula Tiroides/inmunología , Tiroiditis Autoinmune/inmunología , Adolescente , Adulto , Anciano , Antígenos de Superficie/inmunología , Membrana Celular/inmunología , Células Cultivadas , Femenino , Hepatitis Crónica/inmunología , Humanos , Inmunidad Celular/inmunología , Masculino , Persona de Mediana Edad , Mixedema/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
An immunohistochemical technique for the detection of the hepatic asialoglycoprotein receptor (ASGP-R) in cryostat sections of liver by polyclonal and monoclonal anti-ASGP-R antibodies is described. The procedure is based on the alkaline-phosphatase-avidin-biotin complex (ABC-AP) system and important features include fixation of the sections with periodate-lysine-paraformaldehyde (with or without dichromate) and an absolute requirement for blocking of endogenous biotin activity. The sensitivity of the technique is such that binding to ASGP-R can be detected with femtomolar concentrations of monoclonal anti-ASGP-R antibodies and, with polyclonal antisera, approaches that of a radioimmunoassay.
Asunto(s)
Hígado/análisis , Receptores Inmunológicos/análisis , Animales , Anticuerpos , Receptor de Asialoglicoproteína , Avidina/metabolismo , Biotina/metabolismo , Técnicas para Inmunoenzimas , Indicadores y Reactivos/metabolismo , Ratas , Ratas EndogámicasRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Reacciones Cruzadas , Epítopos/inmunología , Lipoproteínas/inmunología , Proteínas de la Membrana , Proteínas/inmunología , Especificidad de la Especie , Animales , Receptor de Asialoglicoproteína , Ensayo de Inmunoadsorción Enzimática , Humanos , Hibridomas/metabolismo , Inmunoglobulinas/biosíntesis , Lipoproteínas/metabolismo , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Proteínas/metabolismo , Conejos , Receptores Inmunológicos/inmunologíaRESUMEN
As part of an investigation into the question of whether virus-induced autoreactivity might contribute to liver damage in viral hepatitis, serial studies (from onset through recovery) of circulating liver autoantibodies have been performed in patients with uncomplicated acute virus A (AVH-A), B (AVH-B) and non-A, non-B (AVH-NANB) hepatitis in whom the time of onset of symptoms could be precisely documented. One hundred and forty-four sera from 35 patients were tested by radioimmunoassay for autoantibodies against the liver-derived lipoprotein complex, LSP, and also against one of its constituents--the asialoglycoprotein receptor, known as hepatic lectin (HL). Anti-LSP antibodies were found in all 10 patients with AVH-A, in 17/18 with AVH-B and in 3/7 with AVH-NANB at titres that declined during recovery. Anti-HL antibodies were detected concurrently in 6 of the AVH-A patients and in 5 with AVH-B but on only 1 occasion in 1 patient with AVH-NANB. Transient cellular immunity to LSP, assayed by a T-lymphocyte migration inhibitory factor test, was detected in 4 of the 6 AVH-B patients tested, 2 of whom also showed concurrent reactivity to HL, but these cellular immune responses did not correlate with production of anti-LSP and/or anti-HL. The findings indicate that humoral immune responses to liver cell surface antigens are frequently triggered by hepatitis A and B viruses, possibly via induction of autoreactive, T-cell independent, liver antigen-specific B lymphocytes. These liver-specific autoreactions have the potential to contribute to hepatocellular damage in virus A and B hepatitis but it seems unlikely that autoimmunity plays a significant pathogenetic role in NANB viral infections.
Asunto(s)
Autoanticuerpos/biosíntesis , Hepatitis Viral Humana/inmunología , Hígado/inmunología , Proteínas de la Membrana , Adulto , Receptor de Asialoglicoproteína , Femenino , Hepatitis A/inmunología , Hepatitis B/inmunología , Hepatitis C/inmunología , Humanos , Técnicas In Vitro , Factores Inhibidores de la Migración de Leucocitos/biosíntesis , Masculino , Proteínas/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunologíaRESUMEN
By means of an indirect T-lymphocyte migration inhibitory factor assay, T-cell sensitisation to the asialoglycoprotein receptor, a liver-specific protein on the surface of hepatocytes, was found in patients with autoimmune chronic active hepatitis (CAH) but not in healthy relatives or spouses. Cellular immunity to a liver-derived lipoprotein complex (LSP) which also contains the asialoglycoprotein receptor, and to which immune reactions have been previously demonstrated in patients with CAH, was also examined. In contrast to the findings with the purified asialoglycoprotein receptor, cell-mediated sensitisation to LSP was detected in all the patients and also in 24% of first-degree relatives and 27% of spouses, suggesting an environmental influence. There was a functional defect in suppressor T lymphocytes specific for liver-derived antigens in 50% of first-degree relatives and 43% of second-degree relatives, but in only 9% of spouses. This abnormality of immunoregulation may be genetically determined and crucial to the development of the disease.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Hepatitis Crónica/inmunología , Hígado/inmunología , Proteínas de la Membrana , Proteínas/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Receptor de Asialoglicoproteína , Enfermedades Autoinmunes/genética , Antígenos HLA/análisis , Hepatitis Crónica/genética , Humanos , Inmunidad Celular , Factores Inhibidores de la Migración de Leucocitos/análisis , Lipoproteínas/inmunología , Masculino , Persona de Mediana Edad , Linaje , Receptores Inmunológicos/inmunologíaRESUMEN
Circulating autoantibodies reacting with affinity-purified, hepatic asialoglycoprotein receptor protein, hepatic lectin (HL), were detected by radioimmunoassay in 15 (83%) of 18 patients with autoimmune chronic active hepatitis (AI-CAH) who had active disease, at titres that showed a positive correlation (P less than 0.05) with severity of periportal inflammation assessed histologically. In contrast, 10 AI-CAH patients whose disease was in remission were all anti-HL seronegative. Anti-HL was also detected in 16 (73%) of 22 patients with hepatitis B virus-related CAH-a similar frequency to that in active AI-CAH but at significantly lower (P less than 0.005) titres. Only 1 of 8 patients with chronic active liver disease due to presumed non-A, non-B (NANB) viral infection and 5 (22%) of 23 with primary biliary cirrhosis were anti-HL seropositive (P less than 0.001 vs active AI-CAH and HBV-CAH) and there was no correlation with severity of periportal inflammation. Anti-HL antibodies were also found in sera from 7 (35%) of 20 patients with acute virus B hepatitis (AVH-B) but were not detected in 10 patients with AVH-A nor in 12 with AVH due to presumed NANB infection. Anti-HL was not found in sera of 12 patients with autoimmune thyroid disease. Hepatic lectin, a highly purifiable, liver-specific cell surface component, by analogy with the acetylcholine and thyrotropin receptors which are, respectively, targets of the pathogenetically-related autoimmune reactions in myasthenia gravis and autoimmune thyroid disease, may be an important target of autoreactions in liver disease.
Asunto(s)
Autoanticuerpos/inmunología , Hepatitis Crónica/inmunología , Hepatitis Viral Humana/inmunología , Receptores Inmunológicos/inmunología , Enfermedad Aguda , Adolescente , Adulto , Anciano , Receptor de Asialoglicoproteína , Enfermedad Crónica , Femenino , Hepatitis B/inmunología , Hepatitis Crónica/sangre , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Masculino , Persona de Mediana EdadRESUMEN
Previous reports have suggested that circulating antibodies reacting with species cross-reactive determinants in the liver-specific membrane lipoprotein (LSP) complex are rare in acute viral hepatitis (AVH) and that their high frequency in chronic active hepatitis (CAH) indicates a fundamental change in antigen specificity of anti-LSP autoantibodies associated with progression to chronic liver disease. In the present study, sera from 20 patients with uncomplicated AVH (10 with virus A and 10 with virus B infection) were examined by radioimmunoassay for antibodies reacting with human, rabbit and rat LSP. All 20 patients were found to be seropositive for antibodies against all 3 LSP preparations. Titres of anti-human and anti-rat LSP were significantly higher (p less than 0.001) than those of anti-rabbit LSP but a strong linear correlation (p less than 0.005) was found between titres of antibodies against the 3 LSP preparations. Cross-inhibition experiments with sera from 10 patients (5 with AVH-A and 5 with AVH-B) failed to reveal any major differences in antigen specificity between anti-rabbit and anti-rat LSP in 8 but results with the remaining 2 patients suggested that their anti-LSP antibodies were not reacting with the same antigens in the 2 LSP preparations. The results indicate that antibodies reacting with species cross-reactive LSP determinants are a normal occurrence in AVH and suggest that, if anti-LSP antibodies are involved in progression to (and/or development of) CAH, this is more likely to be related to a defect in the control of autoreactivity than to inherent differences in target antigen specificities of these autoantibodies in acute and chronic liver diseases, although the latter possibility cannot be totally excluded.
Asunto(s)
Autoanticuerpos/inmunología , Anticuerpos Antihepatitis/inmunología , Hepatitis Viral Humana/inmunología , Proteínas/inmunología , Animales , Reacciones Cruzadas , Epítopos/análisis , Humanos , Hígado/inmunología , Masculino , Proteínas de la Membrana/inmunología , Proteínas/análisis , Conejos/inmunología , Radioinmunoensayo , Ratas/inmunología , Especificidad de la EspecieRESUMEN
To determine whether circulating antibodies against the liver-specific membrane lipoprotein complex (LSP), which occur in a number of liver disorders, are directed at the hepatic asialoglycoprotein receptor protein (hepatic lectin)--recently shown to be a constituent of LSP--a radioimmunoassay for anti-hepatic lectin antibodies was developed based on the use of protein A in situ on staphylococcal cells to precipitate 125I-labelled rabbit hepatic lectin/anti-lectin immune complexes. The assay was used to test sera from 30 patients (15 anti-LSP-positive and 15 anti-LSP-negative) with autoimmune chronic active hepatitis (CAH), hepatitis B virus-positive CAH or primary biliary cirrhosis. Anti-lectin antibodies (at titres of 1:200 to 1:1600) were found in all 15 anti-LSP-positive sera and were not detected in the 15 anti-LSP-negative patients. Hepatic lectin is a highly purifiable, liver-specific, hepatocellular surface component and the anti-lectin assay (which proved sensitive, reliable and easy to perform) will permit further exploration of the role of autoimmune mechanisms in the pathogenesis of various liver diseases.
Asunto(s)
Autoanticuerpos/análisis , Hepatitis Crónica/inmunología , Hígado/inmunología , Proteínas de la Membrana , Receptor de Asialoglicoproteína , Asialoglicoproteínas/metabolismo , Humanos , Lipoproteínas/inmunología , Cirrosis Hepática Biliar/inmunología , Proteínas/inmunología , Radioinmunoensayo , Receptores Inmunológicos/inmunologíaRESUMEN
The value of serum autoantibodies against the liver-specific membrane lipoprotein (LSP) complex in predicting the outcome of treatment withdrawal was assessed in 22 patients with autoimmune chronic active hepatitis who had been maintained in remission on azathioprine and/or prednisolone. At the start of treatment withdrawal 8 patients had anti-LSP antibodies. Within 7-20 weeks (median 12 weeks) all 8 showed biochemical (aminotransferase greater than 200 IU/l) amd histological (piecemeal necrosis) evidence of reactivation of disease. Of the remaining 14 patients, who did not have anti-LSP antibodies at the start of treatment withdrawal, 8 became seropositive within 2-13 weeks (median 6 weeks) and all 8 had relapsed by 21 weeks. In 7 of these eight, anti-LSP first appeared 3 to 10 weeks (median 6 weeks) before and, in the eighth, coincident with biochemical evidence of relapse. The remaining 6 patients continued to be anti-LSP negative and showed no evidence of reactivation of disease throughout the study period (median 29 weeks, range 26-32 weeks). The results show that the presence of anti-LSP antibodies in patients who are apparently in complete remission is invariably associated with reactivation of disease during treatment withdrawal.