AMERICAN JOURNAL OF CLINICAL PATHOLOGY Editorial
The Rocky Road to Discovery
This technique extends the number of methods that can be used to detect anti-mitochondrial antibodies (AMA). The assays in more common use include immunoblotting, 2 enzyme-linked immunoassay,2'3 and traditional immunofluorescence, often using the HEp-2 cell line.3 The specificity and sensitivity of AMA for PBC may approximate 90% but remain somewhat uncertain because (1) clinicians may be reluctant to diagnose PBC in the absence of a positive AMA and (2) liver histopathologic results may not be conclusive. Furthermore, there is considerable laboratory-to-laboratory variation in interpreting AMA, at least using immunofluorescence. Despite all of this, it is commonly accepted that almost all patients with PBC test positive for serum AMAs and that false-positive results are not common in the general population. Indeed, a positive result for AMA in the absence of PBC suggests the presence of another autoimmune or hepatic disorder or a family with an increased predilection for PBC.2'4'5 It is noteworthy that the specific targets of most AMAs have been identified as part of the PDH enzyme complex. Anti-mitochondrial antibodies recognize several inner membrane proteins, designated M2, with molecular weights from 48 to 74 kD. These proteins appear to comprise parts of the PDH enzyme complex with antibodies to El and E2; and protein X is prominent in PBC.6'7 Target proteins may be purified or produced via cloned expression vectors and used in immunoassays such as enzyme-linked immunosorbent assay or immunoblotting. 161
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
More uniformly reproducible measurements with purified components and less laboratory-to-laboratory variation would be expected. Although it is clear that antibodies to PDH inhibit PDH in vitro, it is less certain that they do so in v/vo. 8 AntiPDE seems to have no effect on intact mitochondria in intact cells. Thus the relationship of AMA to the pathogenesis of PBC remains enigmatic. Pyruvate dehydrogenase plays a pivotal role in most cells by converting pyruvate to acetyl-CoA for production of energy.7 The localization of biliary damage cannot be accounted for by enzyme localization because PDH is present throughout the body and is not confined to the biliary tree or the liver. There is one report of an antigen that is cross-reactive with PDH on the surface of cells in PBC but not in healthy individuals.9 It will be important for other investigators to confirm this finding because it could explain the stimulus for AMA and localization of disease to the liver. Of course, there are now several autoantibodies, particularly to the so-called extractable nuclear antigens, which react with intracellular structures (see Miller and associates10 for review). As is the case for anti-PDH in primary biliary cirrhosis, the role of anti-extractable nuclear antigens in disease is unclear. These notions lead us to reconsider the origin of autoimmunity in general and of autoantibodies in particular. The key events leading to a loss of tolerance and the development of autoantibodies or autocellular responses seem to be focused at a trimolecular, or ternary, complex consisting of antigen, the T-cell receptor, and glycoproteins of the major histocompatibility complex (MHC)." Within the thymus, immunologically competent cells are selected to be destroyed (negative selection) or to be exported into the circulation (positive selection) through the interactions at the ternary complex. In this way, anti-self lymphocytes are deleted from the peripheral immune repertoire. If anti-self cells escape this gatekeeping process, there appears to be another mechanism, also through the ternary complex, that renders a potentially active lymphocyte, inactive or anergic.12 The role of the genetically determined MHC in presenting antigen helps explain, in part, the familial or hereditary relationships of some autoimmune states with specific MHC antigens. It also may account for the association of specific MHC antigens with certain microorganisms that presumably contribute
The article in this issue of the American Journal of Clinical Pathology by Elkhalifa and colleagues1 describes a novel method of detecting autoantibodies in primary biliary cirrhosis (PBC). Semipurified components of a pyruvate dehydrogenase (PDH) enzyme ensemble are bound to polystyrene beads before the complex is incubated with patient serum and then fluorescein-labeled anti-human immunoglobulin. The complex is read out by flow cytometric analysis and the intensity of fluorescence related to anti-enzyme antibody titer. Results obtained by flow cytometric analysis closely approximated those obtained by immunoblotting. The data suggest that the intensity of fluorescence was greater for those in the later histopathologic stages of PBC. The results obtained from immunoblotting indicated that samples from subjects in the later stages of disease were more likely to have strong reactions with the PDH components, El alpha and protein X.
162
AMERICAN JOURNAL OF CLINICAL PATHOLOGY Editorial
The available information about the immunopathogenesis of PBC remains rather sparse. The remarkable association of AMA with this disease is indicative that they may play a significant role rather than exist as a diagnostically convenient epiphenomenon. The continuing advancement of knowledge about tolerance and the breakage of tolerance is likely to add insights into the origin of PBC and other autoimmune disorders. Whatever the role, if any, of AMA in PBC, there remains an immediate need to measure these autoantibodies. Like all assays in the clinical laboratory, we expect these determinations to have great specificity, sensitivity, and little within-laboratory or between-laboratory variation. A gold standard is needed. We need to work with clinicians and epidemiologists to develop predictive value models. Finally, we need to be able to test samples as economically as possible. It is reasonable to assume that production of purer antigens will lead to improved reproducibility and perhaps economy of the AMA assay.
This, in turn, should lead to cooperative studies to reevaluate the sensitivity and specificity of AMA and their newly defined subunits. It is probable that clinical subsets of PBC also will be identified. Thus advancements in the basic sciences are haltingly and pain-stakingly integrated into clinical practice and it is by this curious process that we ultimately expect to achieve improved prevention, diagnosis, and management of patients with complex diseases. RUSSELL H. TOMAR, M.D.
University of Wisconsin Hospital and Clinics Madison, Wisconsin REFERENCES 1. Elkhalifa M, Kiechle F, Gordon S, Chen J, Poulik M. Aflowcytometric method for the detection of anti-pyruvate dehydrogenase antibody in primary biliary cirrhosis. Am J Clin Pathol 1991 ;97: 202-208. 2. Van de Water J, Cooper A, Surh C, et al. Detection of autoantibodies to recombinant mitochondrial proteins in patients with primary biliary cirrhosis. N Engl J Med 1989;320:1377-1380. 3. Sundin U. Antibody binding and inhibition of pyruvate dehydrogenase (PDH) in sera from patients with primary biliary cirrhosis. Clin Exp Immunol 1990;81:238-243. 4. Kaplan M. Primary biliary cirrhosis. N Engl J Med 1987;316:521528. 5. Munoz L, Thomas H, Scheuer P, Doniach D, Sherlock S. Is mitochondrial antibody diagnostic of primary biliary cirrhosis? Gut 1981;22:136-140. 6. Gershwin E, Mackay I. Primary biliary cirrhosis: Paradigm or paradox for autoimmunity. Gastroenterology 1991;100:822-833. 7. Patel M, Roche T. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J 1990;4:3223-3233. 8. Van de Water, Surh C, Leung P, et al. Molecular definitions, autoepitopes, and enzymatic activities of the mitochondrial autoantigens of primary biliary cirrhosis. Seminars in Liver Disease 1989;9:132-137. 9. Ghadiminejad I, Baum H. Evidence of the cell-surface localization of antigen cross-reacting with the mitochondrial antibodies of primary biliary cirrhosis. Hepatology 1987;8:743-749. 10. Tan EM: Autoantibodies in pathology and cell biology. Cell 1991; 67:841-842. 11. Sinha A, Lopez M, McDevitt H. Autoimmune diseases: The failure of self tolerance. Science 1990;248:1380-1388. 12. Kronenberg M. Self-tolerance and autoimmunity. Cell 1991;65:537542.
A.J.C.P. • February 1992
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
antigens to the ternary complex. One such association might be HLA B27 with Shigella in Reiter's syndrome. Thus, certain exogenous antigens may lead to autoreactions in individuals who have certain MHC pedigrees. However, as in most areas of human biology, there appear to be safeguards, perhaps at multiple levels. I have commented on the potential to anergize previously reactive lymphocytes. We also might suppose that exogenous antigen could be removed from the circulation before significant exposure to the appropriate MHC. Even after the exposure, the number of autoreactive cells may be too few or in the "wrong" compartment to lead to significant tissue impairment. Suppressor cells or antibodies against an autoantibody (anti-idiotypes) may be present to counteract the effects of the offending elements. Undoubtedly, there are myriad co-factors, such as sex hormones, corticosteroids, and cytokines, that influence these occurrences in ways that are poorly understood. Indeed, the genetic programming of these various factors may explain the involvement of multiple genes associated with individual autoimmune states.
The Rocky Road to Discovery
This technique extends the number of methods that can be used to detect anti-mitochondrial antibodies (AMA). The assays in more common use include immunoblotting, 2 enzyme-linked immunoassay,2'3 and traditional immunofluorescence, often using the HEp-2 cell line.3 The specificity and sensitivity of AMA for PBC may approximate 90% but remain somewhat uncertain because (1) clinicians may be reluctant to diagnose PBC in the absence of a positive AMA and (2) liver histopathologic results may not be conclusive. Furthermore, there is considerable laboratory-to-laboratory variation in interpreting AMA, at least using immunofluorescence. Despite all of this, it is commonly accepted that almost all patients with PBC test positive for serum AMAs and that false-positive results are not common in the general population. Indeed, a positive result for AMA in the absence of PBC suggests the presence of another autoimmune or hepatic disorder or a family with an increased predilection for PBC.2'4'5 It is noteworthy that the specific targets of most AMAs have been identified as part of the PDH enzyme complex. Anti-mitochondrial antibodies recognize several inner membrane proteins, designated M2, with molecular weights from 48 to 74 kD. These proteins appear to comprise parts of the PDH enzyme complex with antibodies to El and E2; and protein X is prominent in PBC.6'7 Target proteins may be purified or produced via cloned expression vectors and used in immunoassays such as enzyme-linked immunosorbent assay or immunoblotting. 161
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
More uniformly reproducible measurements with purified components and less laboratory-to-laboratory variation would be expected. Although it is clear that antibodies to PDH inhibit PDH in vitro, it is less certain that they do so in v/vo. 8 AntiPDE seems to have no effect on intact mitochondria in intact cells. Thus the relationship of AMA to the pathogenesis of PBC remains enigmatic. Pyruvate dehydrogenase plays a pivotal role in most cells by converting pyruvate to acetyl-CoA for production of energy.7 The localization of biliary damage cannot be accounted for by enzyme localization because PDH is present throughout the body and is not confined to the biliary tree or the liver. There is one report of an antigen that is cross-reactive with PDH on the surface of cells in PBC but not in healthy individuals.9 It will be important for other investigators to confirm this finding because it could explain the stimulus for AMA and localization of disease to the liver. Of course, there are now several autoantibodies, particularly to the so-called extractable nuclear antigens, which react with intracellular structures (see Miller and associates10 for review). As is the case for anti-PDH in primary biliary cirrhosis, the role of anti-extractable nuclear antigens in disease is unclear. These notions lead us to reconsider the origin of autoimmunity in general and of autoantibodies in particular. The key events leading to a loss of tolerance and the development of autoantibodies or autocellular responses seem to be focused at a trimolecular, or ternary, complex consisting of antigen, the T-cell receptor, and glycoproteins of the major histocompatibility complex (MHC)." Within the thymus, immunologically competent cells are selected to be destroyed (negative selection) or to be exported into the circulation (positive selection) through the interactions at the ternary complex. In this way, anti-self lymphocytes are deleted from the peripheral immune repertoire. If anti-self cells escape this gatekeeping process, there appears to be another mechanism, also through the ternary complex, that renders a potentially active lymphocyte, inactive or anergic.12 The role of the genetically determined MHC in presenting antigen helps explain, in part, the familial or hereditary relationships of some autoimmune states with specific MHC antigens. It also may account for the association of specific MHC antigens with certain microorganisms that presumably contribute
The article in this issue of the American Journal of Clinical Pathology by Elkhalifa and colleagues1 describes a novel method of detecting autoantibodies in primary biliary cirrhosis (PBC). Semipurified components of a pyruvate dehydrogenase (PDH) enzyme ensemble are bound to polystyrene beads before the complex is incubated with patient serum and then fluorescein-labeled anti-human immunoglobulin. The complex is read out by flow cytometric analysis and the intensity of fluorescence related to anti-enzyme antibody titer. Results obtained by flow cytometric analysis closely approximated those obtained by immunoblotting. The data suggest that the intensity of fluorescence was greater for those in the later histopathologic stages of PBC. The results obtained from immunoblotting indicated that samples from subjects in the later stages of disease were more likely to have strong reactions with the PDH components, El alpha and protein X.
162
AMERICAN JOURNAL OF CLINICAL PATHOLOGY Editorial
The available information about the immunopathogenesis of PBC remains rather sparse. The remarkable association of AMA with this disease is indicative that they may play a significant role rather than exist as a diagnostically convenient epiphenomenon. The continuing advancement of knowledge about tolerance and the breakage of tolerance is likely to add insights into the origin of PBC and other autoimmune disorders. Whatever the role, if any, of AMA in PBC, there remains an immediate need to measure these autoantibodies. Like all assays in the clinical laboratory, we expect these determinations to have great specificity, sensitivity, and little within-laboratory or between-laboratory variation. A gold standard is needed. We need to work with clinicians and epidemiologists to develop predictive value models. Finally, we need to be able to test samples as economically as possible. It is reasonable to assume that production of purer antigens will lead to improved reproducibility and perhaps economy of the AMA assay.
This, in turn, should lead to cooperative studies to reevaluate the sensitivity and specificity of AMA and their newly defined subunits. It is probable that clinical subsets of PBC also will be identified. Thus advancements in the basic sciences are haltingly and pain-stakingly integrated into clinical practice and it is by this curious process that we ultimately expect to achieve improved prevention, diagnosis, and management of patients with complex diseases. RUSSELL H. TOMAR, M.D.
University of Wisconsin Hospital and Clinics Madison, Wisconsin REFERENCES 1. Elkhalifa M, Kiechle F, Gordon S, Chen J, Poulik M. Aflowcytometric method for the detection of anti-pyruvate dehydrogenase antibody in primary biliary cirrhosis. Am J Clin Pathol 1991 ;97: 202-208. 2. Van de Water J, Cooper A, Surh C, et al. Detection of autoantibodies to recombinant mitochondrial proteins in patients with primary biliary cirrhosis. N Engl J Med 1989;320:1377-1380. 3. Sundin U. Antibody binding and inhibition of pyruvate dehydrogenase (PDH) in sera from patients with primary biliary cirrhosis. Clin Exp Immunol 1990;81:238-243. 4. Kaplan M. Primary biliary cirrhosis. N Engl J Med 1987;316:521528. 5. Munoz L, Thomas H, Scheuer P, Doniach D, Sherlock S. Is mitochondrial antibody diagnostic of primary biliary cirrhosis? Gut 1981;22:136-140. 6. Gershwin E, Mackay I. Primary biliary cirrhosis: Paradigm or paradox for autoimmunity. Gastroenterology 1991;100:822-833. 7. Patel M, Roche T. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J 1990;4:3223-3233. 8. Van de Water, Surh C, Leung P, et al. Molecular definitions, autoepitopes, and enzymatic activities of the mitochondrial autoantigens of primary biliary cirrhosis. Seminars in Liver Disease 1989;9:132-137. 9. Ghadiminejad I, Baum H. Evidence of the cell-surface localization of antigen cross-reacting with the mitochondrial antibodies of primary biliary cirrhosis. Hepatology 1987;8:743-749. 10. Tan EM: Autoantibodies in pathology and cell biology. Cell 1991; 67:841-842. 11. Sinha A, Lopez M, McDevitt H. Autoimmune diseases: The failure of self tolerance. Science 1990;248:1380-1388. 12. Kronenberg M. Self-tolerance and autoimmunity. Cell 1991;65:537542.
A.J.C.P. • February 1992
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
antigens to the ternary complex. One such association might be HLA B27 with Shigella in Reiter's syndrome. Thus, certain exogenous antigens may lead to autoreactions in individuals who have certain MHC pedigrees. However, as in most areas of human biology, there appear to be safeguards, perhaps at multiple levels. I have commented on the potential to anergize previously reactive lymphocytes. We also might suppose that exogenous antigen could be removed from the circulation before significant exposure to the appropriate MHC. Even after the exposure, the number of autoreactive cells may be too few or in the "wrong" compartment to lead to significant tissue impairment. Suppressor cells or antibodies against an autoantibody (anti-idiotypes) may be present to counteract the effects of the offending elements. Undoubtedly, there are myriad co-factors, such as sex hormones, corticosteroids, and cytokines, that influence these occurrences in ways that are poorly understood. Indeed, the genetic programming of these various factors may explain the involvement of multiple genes associated with individual autoimmune states.
