Anti-neutrophil cytoplasmic antibodies in inflammatory bowel disease: do they recognize different subsets of a heterogeneous disease?

OBJECTIVE: To evaluate the prevalence of anti-neutrophil cytoplasmic antibodies in a series of patients with inflammatory bowel disease, the discriminatory value of these antibodies in differentiating between ulcerative colitis and Crohn's disease, their antigen specificity and their correlation with epidemiological and clinical variables. METHODS: Serum anti-neutrophil cytoplasmic antibodies were evaluated by indirect immunofluorescence and immunoblotting using neutrophils isolated from peripheral blood and by enzyme-linked immunosorbent assays (ELISAs) using proteinase 3 and myeloperoxidase as antigens. RESULTS: Anti-neutrophil cytoplasmic antibodies were detected by immunofluorescence in 43 (39.8%) of 108 patients with ulcerative colitis, in 11 (11.9%) of 92 patients with Crohn's disease (P < 0.001) and 5 (6.8%) of 73 control patients. The predominant pattern was perinuclear staining around neutrophil nuclei (44 of 59, 75%); a homogeneous cytoplasmic staining was present in 15 (25%) of 59 sera, mainly among Crohn's disease and control patients. The ELISAs gave no positive results. Recognition of proteins of relative molecular masses 27,000 and 49,000 at immunoblotting was common to ulcerative colitis, Crohn's disease and control sera. The proteins of relative molecular masses 32,000 and 106,000 were recognized exclusively by 11% of anti-neutrophil-positive ulcerative colitis sera. No significant correlation was found between the presence of anti-neutrophil cytoplasmic antibodies and the demographic and clinical characteristics of the patients. CONCLUSION: Anti-neutrophil cytoplasmic antibodies are detectable in a large proportion of patients with ulcerative colitis, but their prevalence in a limited proportion of patients with Crohn's disease reduces their discriminatory capability. The persistence of anti-neutrophil cytoplasmic antibodies after total colectomy and the absence of a correlation between the activity of the disease and the presence or titre of these antibodies support the hypothesis that anti-neutrophil cytoplasmic antibodies are not simply an epiphenomenon of colonic inflammation.

Detection of antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis: a comparison of the alkaline phosphatase and immunofluorescent techniques.

BACKGROUND: The reported prevalence of antineutrophil cytoplasmic antibodies (ANCA) in primary sclerosing cholangitis (PSC) varies considerably (26-85%). Part of this may reflect methodological differences but part may reflect the differences in the patient groups analysed. To resolve this issue we compared the sensitivity and specificity of the immunoalkaline phosphatase (IALP) and immunofluorescence (IF) techniques in four different populations. METHOD: Sera from four centres were tested blind on alcohol-fixed neutrophils using both techniques. PATIENTS: USA: 14 PSC, 14 primary biliary cirrhosis (PBC); Sweden: 32 PSC, 3 autoimmune hepatitis (AIH), 14 PBC, 11 chronic liver disease; Norway: 32 PSC, 14 AIH, 13 PBC, 1 hepatitis C. Italy: 8 PSC, 14 PBC, 8 viral hepatitis. Thirty-six normal healthy volunteers from Oxford, together with positive and negative controls, were also tested. RESULTS: The healthy controls were all ANCA negative. The diagnostic sensitivity and specificity, respectively, of ANCA for PSC using the IALP technique for the different test sera were: USA 71% and 93%, Sweden 66% and 96%, Norway 69% and 46%, Italy 50% and 95%. The diagnostic sensitivity and specificity, respectively, of the IF technique on the same sera were: USA 50% and 86%, Sweden 56% and 86%, Norway 47% and 61%, Italy 50% and 91%. Overall, combining all four groups, detection of ANCA using the IALP technique gave a diagnostic sensitivity of 66% with a specificity of 74% for PSC. In contrast, the IF technique gave an overall diagnostic of only 51% (P = 0.044, compared with IALP) with a specificity of 73%. Although overall the IALP technique was more sensitive than IF, the differences in sensitivity and specificity between the two techniques did not reach statistical significance for any individual group. Furthermore, the small differences in sensitivity between the four groups using either technique were not significant. However, the IALP technique had greater specificity in the US, Swedish and Italian groups compared with the Norwegian group (P < 0.05) whereas no statistically significant differences in specificity were noted between the groups using the IF technique. CONCLUSION: This study shows that the IALP method of ANCA detection is at least as sensitive as IF for the serological diagnosis of PSC. Indeed, combining data from all four centres, the IALP technique was significantly more sensitive than IF. We therefore recommend the use of the IALP technique, which is also easier to interpret and does not require the use of a specialist fluorescent microscope. The lack of a wide variation in sensitivity between IALP and IF for any individual patient group reported in this study suggests that the previously reported regional differences in ANCA prevalence in PSC of between 26% and 85% may be patient, related, rather than due to ethnic or methodological differences in ANCA detection, perhaps reflecting possible disease heterogeneity within PSC, or case selection bias. Further studies are needed to investigate this intriguing possibility. Such differences, if confirmed, will need to be taken into account when assessing the use of ANCA as a serological marker of PSC.

Characterization of antimitochondrial antibodies in health adults.

The detection of antimitochondrial antibodies (AMAs) is an important criterion for the diagnosis of primary biliary cirrhosis (PBC). During the last decade, the mitochondrial autoantigens have been cloned, sequenced, and identified as members of the 2-oxo-acid dehydrogenase pathway, including the E2 subunits of pyruvate dehydrogenase (PDC-E2), branched-chain 2-oxo-acid dehydrogenase (BCOADC-E2), and 2-oxo-glutarate dehydrogenase (OGDC-E2). We have developed a rapid and sensitive diagnostic test for use in PBC based on a triple hybrid recombinant molecule (r-MIT3) that contains the autoepitopes of PDC-E2, BCOADC-E2, and OGDC-E2. To help understand the frequency and antigen specificity of AMAs in an asymptomatic population and to identify patients with early disease, we investigated the prevalence of AMA, by enzyme-linked immunosorbent assay (ELISA), in a cohort of 1,530 people from northern Italy. Positive sera were further analyzed for immunoglobulin (Ig) isotypes, subclasses, and epitopes of AMA by a combination of ELISA and immunoblotting. In this cohort of 1,530 people, 9 (0.5%) reacted to r-MIT3 by ELISA. Of the 9 reactive sera, 2 recognized PDC-E2, 2 of 9 recognized BCOADC-E2, 1 of 9 recognized OGDC-E2, 2 of 9 recognized both PDC-E2 and BCOADC-E2, and 1 of 9 recognized PDC-E2 and OGDC-E2. AMA reactivity was primarily IgM and IgA. Epitope mapping revealed an AMA pattern of reactivity to PDC-E2 that differed from that found in patients with histologically proven PBC in most of the sera. However, 1 sera of a 72-year-old female with a normal alkaline phosphatase had an AMA profile identical to typical PBC. After a variable follow-up period (8-14 months), sera from 8 of 9 of these people were re-obtained for AMA and relative epitope mapping. Interestingly, the reactivity had a wider AMA pattern than before.

Persistence of autoantibodies against recombinant mitochondrial and nuclear pore proteins after orthotopic liver transplantation for primary biliary cirrhosis.

Primary biliary cirrhosis is an autoimmune disease characterized by high titer autoantibodies predominantly against mitochondrial antigens PDC-E2, BCOADC-E2 and OGDC-E2. Currently orthotopic liver transplant (OLT) is the major form of treatment for end-stage primary biliary cirrhosis (PBC), but it is still unclear whether the autoimmune response continues post-transplantation. In this study we took advantage of a well-defined collection of sera collected serially before and after liver transplantation. We assayed these sera for quantitative and isotype-specific titers of antibodies against a set of recombinant mitochondrial autoantigens. We also studied reactivity to gp210. Serum samples were taken before transplantation and at intervals of 6 months, 1, 2, and 3 years after OLT. Before OLT 24/35 patients were AMA-positive, including seven out of the 35 to PDC-E2 alone, eight to both PDC-E2 and OGDC-E2, six to both PDC-E2 and BCOADC-E2, two to BCOADC-E2 alone and one to OGDC-E2. Following OLT, the frequency of sera that responded to PDC-E2 alone increased from seven to 12/35. Similarly, reactivity to BCOADC-E2 slightly increased from two to four out of 35. However, there was an overall decrease in sera that responded to more than one antigen. Neither Ig isotype nor subclass of the autoimmune response changed following OLT. Findings with gp210 were similar, in that reactivity to gp210 was found in nine out of 35 patients pre-OLT; following OLT the frequency decreased to seven out of 35 patients. Overall, the titers of AMAs decline slightly during the first year post-OLT, but are equivalent to pre-OLT values by 6 months. Moreover, the antibody subclass/ isotype remained unchanged. These data suggest that the removal of a diseased PBC liver has little, if any, impact on the serological characteristics of PBC. Moreover, it provides information regarding the natural history of PBC, particularly on the long latency time for disease development.