å¡ ORIGINAL
ARTICLE
å¡
Anti-Centromere Antibody and CREST Syndrome in Patients with Primary Biliary Cirrhosis Isao Shoji, Tohru Takagi and Reiji Kasukawa Anti-centromere antibodies (ACA) in 41 sera from patients with primary biliary cirrhosis (PBC) were analyzed by an immunoblotting method and the correlation between the presence ofACA and the clinical features in these PBC patients was studied. In 10 of 16 ACA-positive PBC patients, one or more clinical features of CREST syndrome (PBC-CREST) were found. Statistical differences were observed in age at disease onset, serum levels of IgM and total bilirubin and titer of anti-M2 antibody, between PBC-CREST patients and the PBC patients without CREST symptoms (PBC-non CREST). By immunoblotting analysis, three major epitopes of ACA were identified at 18 kD, 80 kD and 140 kD polypeptides. The 18 kD polypeptides were detected in all 16 ACA-positive PBC patients. From these results, it is suggested that ACA-positive PBC-CREST (Internal Medicine 31:1348- 1355, 1992) patients can be separated from ACA-negative PBC-CREST and PBC-non CREST patients. Key words: autoimmune liver disease, autoantibody, epitope, crude centromere protein, immunoblotting analysis
Introduction Primary biliary cirrhosis (PBC), which was first des cribed in 1950 (1), is an enigmatic chronic autoimmune liver disease (2) with progressive inflammatory obliter ation of the intrahepatic bile ducts and autoantibody to mitochondria (AMA) (3, 4). The major antigens of mitochondria, termed M2 (5), have been identified as E2 components of pyruvate dehydrogenase (PDH) Patients PBC(PDC) have been reported to have some enzyme with complex (6, 7). incidence of other autoimmune disorders such as Sjogren's syndrome, Hashimoto's disease, progressive systemic sclerosis (PSS), and CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) syndrome (8, 9). Anti-centromere antibody (ACA) was first reported in sera of patients with PSS (10) and subsequently recognized more fre Three majory centromere antigens have been recognized quently in sera of patients with CREST syndrome (ll). using human cell lines and a chromosomal preparation, and designated as centromere protein A (CENP-A, 17kD polypeptide), centromere protein B (CENP-B, 80 kD polypeptide) , and centromere protein C (CENP-C, 140 kD polypeptide) (12).
In this study, we attempted to identify the major epitope of ACA in sera from patients with PBC and to clarify the correlation between the presence of ACA epitopes and the clinical in patients with PBC. Using purified PDH and afeatures preparation of centromere enriched chromosome of HeLa cells' nuclei, the reactivity of sera from patients with PBC was analyzed for epitope specificity of these antigens. The serological results obtained were compared with clinical features of CREST syndrome in PBC. Materials and Methods
Patien ts Forty-one patients with PBC were studied. Diagnosis of PBC was based on one of the following diagnostic criteria (13): 1) patients with pathohistological features showing chronic non-suppurative distructive cholangitis (CNSDC). 2) patients positive for AMA with patho histological changes compatible with PBC. 3) patients highly suspicious of PBC due to positive AMA, clinical symptoms and clinical course despite a lack of patho histological features. Sera from these patients were collected at the Department of Internal Medicine II of College, Fukushima Medical College Hospital and affiliated From the Department of Internal Medicine II, Fukushima Medical Fukushima Received for publication April 16, 1992; Accepted for publication October 12, 1992 Reprint requests should be addressed to Dr. Isao Shoji, the Department of Internal Medicine II, Fukushima Medical College, Hikarigaok Fukushima, 960-12, Japan 1348
Internal
Medicine
Vol. 31, No. 12 (December
1992)
ACA and CREST Syndrome in PBC hospitals. Findings correlating to CREST syndrome were diagnosed as follows: calcinosis on X-ray film of extremities. Raynaud's phenomenon on history, esophageal dysmotility on barium examination sclero dactyly and telangiectasia on objective observation. A diagnosis of CREST syndrome was established when all of these five features were present and that of incomplete CREST syndrome was established when at least three out of five features were present. In this report, PBC patients who had at least one or more features of CREST syndrome were designated as PBC-CREST patients and the other PBC patients were designated as PBC-non CREST patients. A diagnosis of Sjogren's syndrome was made when typical symptoms of dry mouth or dry eyes and aofpositive Shirmer's testorwere observed. Other Diagnosis either symptomatic asymptomatic autoimmune diseases according were diagnosed the criteria PBC was determined to the by slightly modified for each disease. criteria proposed by Long et al (14), Roll et al (15), and Sasaki et al (13); patients without itching and icterus or patients who were diagnosed by chance were defined as asymptomatic PBC. Detection of anti-mitochondrial antibody (AMA) and anti-centromere antibody (A CA) AMA were detected by an indirect immunofluore scence test with sections of rat kidney. Titers of AMA were measured by using an enzyme-linked immunosorbent assay (EIA) kit named MESA cup (MBL Inc, CO., LTD, Nagoya, Japan), which could detect anti-M2 anti body. ACA were also detected by an indirect immuno fluorescence test using HEp-2 cells. The typical discrete speckled pattern of the nucleus in metaphase was con sidered as to be positive for ACA. Purification of PDH PDH were purified from bovine heart by the method of Stanley and Perham (16). Bovine heart was cut into cubes and crushed in a Waring blender for 5 minutes with Triton-X. The homogenate was precipitated sub sequently with polyethylene glycol at two steps and separated by isoelectric precipitation. Each sample of the separated complexes was purified with gel filtration on a column of Sepharose CL-2B. All steps were carried out at 4°C. The preparation obtained was used for immunoblotting analysis. protein (CENP) Preparation of centromere Centromere enriched nuclear protein fractions were prepared from HeLa cell's nuclei according to the modi fied method described by Masumoto et al (17): HeLa cells were treated overnightand withcentrifuged 0.05 /ig/mlatcolcemid. Mitotic cells were collected 600 xg for 5 minutes. The pellets were resuspended in isolating buffer (15mM Tris-HCl, pH 7.4, 2mM EDTA, 0.2mM Internal
Medicine
Vol. 31, No. 12 (December
1992)
EGTA, 0.2mM spermine, 0.5mM spermidine, 80mM KC1, 7mM /?-mercaptoethanol) containing 0.1% digi tonin. The mixture was agitated gently at 4°C for 1 hour. After centrifugation at 600xg for 5 minutes, which was repeated 5 times, the suspended cells were destroyed by sonicator and the procedure was repeated 10 times. The destructed cells were centrifuged and washed with PBS and the supernatants thus obtained were used as a crude centromere protein (CENP) for immunoblotting analysis. Immunoblotting analysis After electrophoreses of these antigenic preparations on SDS-PAGE according to the method described by Laemmli (18), the protein was transfered to polyvinyldene difluoride membrane (PVDM) through a modification of the method described by Towbin et al (19) and the membrane was treated with 3% BSA for 1 hour at room temperature. After washing with PBS, each column of the membrane was cut into longitudinal strips and incubated at room temperature for 1 hour with serum of PBC diluted at 1:500. The strips were washed 5 times for 5 minutes each with PBS-Tween and incubated at room temperature for 1 hour with an ALP-conjugated goat antihuman IgG (Tago Inc, Burlingame, USA) serum diluted at 1:1,000. After washing 5 times for 5 minutes each with PBS-Tween, color was developed with naphtol AS-MX phosphate and fast blue BB salt. Results Forty-one patients with PBC, 35 females and 6 males, were studied. The age at disease onset ranged from 24 to 78 years. AMA were detected in 34 of 41 (83%) patients by indirect immunofluorescence test. ACA were detected in 16 of 41 (39%) patients by indirect immunofluorescence test. The mean age at disease onset was 59 ± 10.4 (mean ± SD) in ACA-positive and The difference was statistically significant 48±12.5 (meanbetween ± SD) inthem ACA-negative patients. (p
ARTICLE
å¡
Anti-Centromere Antibody and CREST Syndrome in Patients with Primary Biliary Cirrhosis Isao Shoji, Tohru Takagi and Reiji Kasukawa Anti-centromere antibodies (ACA) in 41 sera from patients with primary biliary cirrhosis (PBC) were analyzed by an immunoblotting method and the correlation between the presence ofACA and the clinical features in these PBC patients was studied. In 10 of 16 ACA-positive PBC patients, one or more clinical features of CREST syndrome (PBC-CREST) were found. Statistical differences were observed in age at disease onset, serum levels of IgM and total bilirubin and titer of anti-M2 antibody, between PBC-CREST patients and the PBC patients without CREST symptoms (PBC-non CREST). By immunoblotting analysis, three major epitopes of ACA were identified at 18 kD, 80 kD and 140 kD polypeptides. The 18 kD polypeptides were detected in all 16 ACA-positive PBC patients. From these results, it is suggested that ACA-positive PBC-CREST (Internal Medicine 31:1348- 1355, 1992) patients can be separated from ACA-negative PBC-CREST and PBC-non CREST patients. Key words: autoimmune liver disease, autoantibody, epitope, crude centromere protein, immunoblotting analysis
Introduction Primary biliary cirrhosis (PBC), which was first des cribed in 1950 (1), is an enigmatic chronic autoimmune liver disease (2) with progressive inflammatory obliter ation of the intrahepatic bile ducts and autoantibody to mitochondria (AMA) (3, 4). The major antigens of mitochondria, termed M2 (5), have been identified as E2 components of pyruvate dehydrogenase (PDH) Patients PBC(PDC) have been reported to have some enzyme with complex (6, 7). incidence of other autoimmune disorders such as Sjogren's syndrome, Hashimoto's disease, progressive systemic sclerosis (PSS), and CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) syndrome (8, 9). Anti-centromere antibody (ACA) was first reported in sera of patients with PSS (10) and subsequently recognized more fre Three majory centromere antigens have been recognized quently in sera of patients with CREST syndrome (ll). using human cell lines and a chromosomal preparation, and designated as centromere protein A (CENP-A, 17kD polypeptide), centromere protein B (CENP-B, 80 kD polypeptide) , and centromere protein C (CENP-C, 140 kD polypeptide) (12).
In this study, we attempted to identify the major epitope of ACA in sera from patients with PBC and to clarify the correlation between the presence of ACA epitopes and the clinical in patients with PBC. Using purified PDH and afeatures preparation of centromere enriched chromosome of HeLa cells' nuclei, the reactivity of sera from patients with PBC was analyzed for epitope specificity of these antigens. The serological results obtained were compared with clinical features of CREST syndrome in PBC. Materials and Methods
Patien ts Forty-one patients with PBC were studied. Diagnosis of PBC was based on one of the following diagnostic criteria (13): 1) patients with pathohistological features showing chronic non-suppurative distructive cholangitis (CNSDC). 2) patients positive for AMA with patho histological changes compatible with PBC. 3) patients highly suspicious of PBC due to positive AMA, clinical symptoms and clinical course despite a lack of patho histological features. Sera from these patients were collected at the Department of Internal Medicine II of College, Fukushima Medical College Hospital and affiliated From the Department of Internal Medicine II, Fukushima Medical Fukushima Received for publication April 16, 1992; Accepted for publication October 12, 1992 Reprint requests should be addressed to Dr. Isao Shoji, the Department of Internal Medicine II, Fukushima Medical College, Hikarigaok Fukushima, 960-12, Japan 1348
Internal
Medicine
Vol. 31, No. 12 (December
1992)
ACA and CREST Syndrome in PBC hospitals. Findings correlating to CREST syndrome were diagnosed as follows: calcinosis on X-ray film of extremities. Raynaud's phenomenon on history, esophageal dysmotility on barium examination sclero dactyly and telangiectasia on objective observation. A diagnosis of CREST syndrome was established when all of these five features were present and that of incomplete CREST syndrome was established when at least three out of five features were present. In this report, PBC patients who had at least one or more features of CREST syndrome were designated as PBC-CREST patients and the other PBC patients were designated as PBC-non CREST patients. A diagnosis of Sjogren's syndrome was made when typical symptoms of dry mouth or dry eyes and aofpositive Shirmer's testorwere observed. Other Diagnosis either symptomatic asymptomatic autoimmune diseases according were diagnosed the criteria PBC was determined to the by slightly modified for each disease. criteria proposed by Long et al (14), Roll et al (15), and Sasaki et al (13); patients without itching and icterus or patients who were diagnosed by chance were defined as asymptomatic PBC. Detection of anti-mitochondrial antibody (AMA) and anti-centromere antibody (A CA) AMA were detected by an indirect immunofluore scence test with sections of rat kidney. Titers of AMA were measured by using an enzyme-linked immunosorbent assay (EIA) kit named MESA cup (MBL Inc, CO., LTD, Nagoya, Japan), which could detect anti-M2 anti body. ACA were also detected by an indirect immuno fluorescence test using HEp-2 cells. The typical discrete speckled pattern of the nucleus in metaphase was con sidered as to be positive for ACA. Purification of PDH PDH were purified from bovine heart by the method of Stanley and Perham (16). Bovine heart was cut into cubes and crushed in a Waring blender for 5 minutes with Triton-X. The homogenate was precipitated sub sequently with polyethylene glycol at two steps and separated by isoelectric precipitation. Each sample of the separated complexes was purified with gel filtration on a column of Sepharose CL-2B. All steps were carried out at 4°C. The preparation obtained was used for immunoblotting analysis. protein (CENP) Preparation of centromere Centromere enriched nuclear protein fractions were prepared from HeLa cell's nuclei according to the modi fied method described by Masumoto et al (17): HeLa cells were treated overnightand withcentrifuged 0.05 /ig/mlatcolcemid. Mitotic cells were collected 600 xg for 5 minutes. The pellets were resuspended in isolating buffer (15mM Tris-HCl, pH 7.4, 2mM EDTA, 0.2mM Internal
Medicine
Vol. 31, No. 12 (December
1992)
EGTA, 0.2mM spermine, 0.5mM spermidine, 80mM KC1, 7mM /?-mercaptoethanol) containing 0.1% digi tonin. The mixture was agitated gently at 4°C for 1 hour. After centrifugation at 600xg for 5 minutes, which was repeated 5 times, the suspended cells were destroyed by sonicator and the procedure was repeated 10 times. The destructed cells were centrifuged and washed with PBS and the supernatants thus obtained were used as a crude centromere protein (CENP) for immunoblotting analysis. Immunoblotting analysis After electrophoreses of these antigenic preparations on SDS-PAGE according to the method described by Laemmli (18), the protein was transfered to polyvinyldene difluoride membrane (PVDM) through a modification of the method described by Towbin et al (19) and the membrane was treated with 3% BSA for 1 hour at room temperature. After washing with PBS, each column of the membrane was cut into longitudinal strips and incubated at room temperature for 1 hour with serum of PBC diluted at 1:500. The strips were washed 5 times for 5 minutes each with PBS-Tween and incubated at room temperature for 1 hour with an ALP-conjugated goat antihuman IgG (Tago Inc, Burlingame, USA) serum diluted at 1:1,000. After washing 5 times for 5 minutes each with PBS-Tween, color was developed with naphtol AS-MX phosphate and fast blue BB salt. Results Forty-one patients with PBC, 35 females and 6 males, were studied. The age at disease onset ranged from 24 to 78 years. AMA were detected in 34 of 41 (83%) patients by indirect immunofluorescence test. ACA were detected in 16 of 41 (39%) patients by indirect immunofluorescence test. The mean age at disease onset was 59 ± 10.4 (mean ± SD) in ACA-positive and The difference was statistically significant 48±12.5 (meanbetween ± SD) inthem ACA-negative patients. (p
