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Enzymes as autoantigens
779
EDITORIALS
Enzymes as autoantigens Autoimmunity
accounts tor about 80% ot
newly
Addison’s disease.’ This type of adrenal failure can be associated with two distinct clusters of
diagnosed
disorders.2Type 1 polyendocrine autoimmunity and is usually recognised in the coincidence of chronic by mucocutaneous candidosis and hypoparathyroidism; Addison’s disease ensues 5-10 years later. The much commoner type 2 syndrome is the association of at least two of the following conditions: Addison’s disease, autoimmune hypothyroidism, Graves’ disease, type 1 diabetes, and premature ovarian failure. Both syndromes may be accompanied by other autoimmune conditions--eg, vitiligo and pernicious anaemia-but there are several distinctive features. In particular, the type 1 syndrome has an autosomal recessive inheritance, not linked to genes in the HLA-D region; the type 2 syndrome has a dominant inheritance pattern and strong links with HLA-DR3.3-5 Patients with type 2 syndrome present much later than those with type 1. Immunological heterogeneity is suggested by the presence of steroid cell autoantibodies in over 85% of patients with the type 1 syndrome1,6 whereas these antibodies are found in less than 20 % of patients with sporadic or type-2-associated Addison’s disease.6,7 The high frequency of autoantibodies presumably explains why gonadal failure commonly occurs in type 1 syndrome. From immunofluorescence staining patterns, steroid cell antibodies seem to react with several antigens, which are consistently present in the adrenal but have variable distribution in the ovary, testis, and placenta.8 Adrenal autoantibodies that do not cross-react with antigens in other steroid cells are found in about two-thirds of all Addison’s patients (excluding those with type 1 syndrome).1,9 The nature of these shared and unique autoantigens remains unknown. By contrast, the identity of the thyroid microsomal antigen in thyroiditis’O and of beta cell
syndrome
childhood
is
rare
autoantigens
in type 1 diabetes11,12 has
elucidated. The report
by Krohn and colleagues
lately been
in this issue (p 770) begins to redress that imbalance. Using sera from patients with type 1 polyendocrine autoimmunity to screen an expressed fetal adrenal cDNA library, these researchers obtained and sequenced immunoreactive clones; all but 1 corresponded to part of the known sequence of 17a-hydroxylase. This enzyme is essential for testosterone and oestrogen production, so it is highly likely that 17a-hydroxylase is also one of the gonadal steroid cell autoantigens recognised by autoantibodies. The striking negative finding of this paper (albeit made with only two samples) is the lack of reactivity with 17a-hydroxylase of sera from patients with sporadic Addison’s disease. These sera did not even give an immunoprecipitation reaction with adrenal homogenate, probably because the titre of adrenal antibodies was low. This observation suggests that autoantibodies in patients with sporadic and type-2-associated Addison’s disease recognise adrenal antigens other than 17a-hydroxylase. Patients with the type 1 syndrome may likewise have multiple adrenal autoantibodies, since immunoblotting showed that such sera reacted with four separate proteins whose molecular weights differed from that of 17a-hydroxylase. However, further work is needed to exclude the possibility that these proteins were proteolytic fragments of the enzyme. As Krohn et al point out, the 20-week fetal adrenal used to prepare their cDNA library may have been too immature to express the complete adult repertoire of proteins, and other autoantigenic steroid enzymes are now being
sought. Characterisation of the autoantigens has given a fresh impetus to research into the pathogenesis of adrenal failure in Addison’s disease. One possibility is that autoantibodies may impair target organ enzyme function, but this general mechanism remains equivocal even for the best characterised enzyme autoantigen, thyroid peroxidase. 13,11 It is unclear how readily antibodies have access to intracellular enzymes such as 17a-hydroxylase in vivo or how adrenal cell surface autoantigens identified by immunofluorescence are related to those in the cytoplasm.1s Similar difficulties with access may likewise reduce the pathogenic importance of steroid enzyme complement fixation by autoantibodies. If complement-mediated adrenal injury does occur, it could operate through more subtle mechanisms than simple cell lysis-target cells may respond to sublethal complement attack with reduced metabolic responses and release of inflammatory mediators.16 Peripheral blood T-cell responses to thyroid peroxidase and glutamic acid decarboxylase peptides have been described in Hashimoto’s thyroiditis and type 1 diabetes, respectively. 11-19 These findings raise the alternative possibility that production of autoantibodies against
780
enzymes is
secondary phenomenon, the initiating event being T-cell mediated cytotoxicity directed against endogenous, enzyme-derived peptides coexpressed with class I (and even class II) major histocompatibility complex molecules on target cells. This report is the latest in a series showing that previously ill-defined autoantigens turn out to be enzymes. To the list provided by Krohn et al we can add carboxypeptidase-H in diabetes,12 and myeloperoxidase and a 29 kDa neutrophil proteinase in systemic vasculitis.2O,21 These observations should not come as a surprise: we know that many proteins function as enzymes (at least 10 000 in man) and that the distribution of a proportion of these molecules is restricted. Clearly target-organ-specific enzymes should be considered as candidate autoantigens in a
other obscure autoimmune disorders. 1. Irvine
WJ, Barnes EW. Addison’s disease, ovarian failure and
hypoparathyroidism. Clin Endocrinol Metab 1975; 4: 379-434. 2. Neufeld JDN, Maclaren NK, Blizzard RM. Two types of autoimmune Addison’s disease associated with different polyglandular autoimmune (PGA) syndromes. Medicine 1981; 60: 355-62. 3. Ahonen P, Koskimies S, Likki M-L, Tillikainen A, Perheentupa J. The expression of autoimmune polyglandular disease type 1 appears associated with several HLA-A antigens but not with HLA-DR. J Clin Endocrinol Metab 1988; 66: 1151-57. 4. Eisenbarth GS, Jackson RA. Immunogenetics of polyglandular failure and related diseases. In: Farid NR, ed. HLA in endocrine and metabolic disorders. New York: Academic Press, 1981: 235-64. 5. Maclaren NK, Riley WJ. Inherited susceptibility to autoimmune Addison’s disease is linked to human leukocyte antigens-DR3 and/or DR4, except when associated with type 1 autoimmune polyglandular syndrome. J Clin Endocrinol Metab 1986; 62: 455-59. 6. Ahonen P, Miettinen A, Perheentupa J. Adrenal and steroidal cell antibodies in patients with autoimmune polyglandular disease type 1 and risk of adrenocortical and ovarian failure. J Clin Endocrinol Metab 1987; 64: 494-500. 7. Elder M, Maclaren N, Riley W. Gonadal autoantibodies in patients with hypogonadism and/or Addison’s disease. J Clin Endocrinol Metab 1981; 52: 1137-42. 8. Sotsiou F, Bottazzo GF, Doniach D. Immunofluorescence studies on autoantibodies to steroid-producing cells, and to germline cells in endocrine diseases and infertility. Clin Exp Immunol 1980; 39: 97-111. 9. Latinne D, Vandeput Y, De Bruyere M, Bottazzo F, Sokal G, Crabbe J. Addison’s disease: immunological aspects. Tissue Antigens 1987; 30: 23-24. 10. Czarnocka B, Ruf J, Ferrand M, Carayon P, Lissitky S. Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases. FEBS Lett 1985; 190: 147-51. 11. Baekkeskov S, Jan-Aanstoot H, Christgau S, et al. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABAsynthesizing enzyme glutamine acid decarboxylase. Nature 1990; 347: 151-56. 12. Castano L, Russo E, Zhou L, Lipes MA, Eisenbarth GS. Identification and cloning of a granule autoantigen (carboxypeptidase-H) associated with type 1 diabetes. J Clin Endocrinol Metab 1991; 73: 1197-201. 13. Kohno Y, Yamaguchi F, Saito K, Niimi H, Nishikawa T, Hosoya T. Anti-thyroid peroxidase antibodies in sera from healthy subjects and from patients with chronic thyroiditis: differences in the ability to inhibit thyroid peroxidase activities. Clin Exp Immunol 1991; 85: 459-63. 14. Saller B, Hormann R, Mann K. Heterogeneity of autoantibodies against thyroid peroxidase in autoimmune thyroid disease: evidence against antibodies directly inhibiting peroxidase activity as regulatory factors in thyroid hormone metabolism. J Clin Endocrinol Metab 1991; 72: 188-95. 15. Khoury EL, Hammond L, Bottazzo GF, Doniach D. Surface-reactive antibodies to human adrenal cells in Addison’s disease. Clin Exp Immunol 1981; 45: 48-55. 16. Morgan BP. Complement membrane attack on nucleated cells: resistance, recovery and non-lethal effects. Biochem J 1989; 264: 1-14. 17. Dayan CM Londei M, Corcoran AE, et al. Autoantigen recognition by thyroid-infiltrating T cells in Graves’ disease. Proc Natl Acad Sci USA 1991; 88: 7415-19.
18. Tandon N, Freeman M, Weetman AP. T cell responses to synthetic thyroid peroxidase peptides in autoimmune thyroid disease. Clin Exp Immunol 1991; 86: 56-60. 19. Atkinson MA, Kaufman DL, Campbell L, et al. Response of peripheral-blood mononuclear cells to glutamate decarboxylase in insulin-dependent diabetes. Lancet 1992; 339: 458-59. 20. Falk RJ, Jennett JC. Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N EnglJ Med 1988; 318: 1651-57. 21. Ludemann J, Utecht B, Gross WL. Anti-neutrophil cytoplasm antibodies in Wegener’s granulomatosis recognize an elastinolytic enzyme. J Exp Med 1990; 171: 357-62.
Streptokinase plus aspirin does the trick: ISIS-3 The Second International Study of Infarct Survival (ISIS-2) confirmed the benefits of streptokinase thrombolysis in acute myocardial infarction shown in the Italian GISSI-1 trial and also showed enhanced survival when streptokinase was combined with aspirin-thrombolysis quickly became established as standard therapy for heart attacks.1,2 ISIS-3, reported on p 753, had the more difficult task of investigating, first, whether the addition of high-dose subcutaneous heparin to aspirin conferred any additional benefit and whether recombinant second, tissue-type activator or plasminogen (duteplase) anisoylated plasminogen streptokinase activator complex (anistreplase) was any better than streptokinase. The clearest message to emerge from this massive enterprise (41 299 patients randomised to different thrombolytic regimens) is that both null hypotheses survived intact-ie, no thrombolytic regimen performed better, in terms of 35-day survival, than the simple and cheap ISIS-2 cocktail. Use of subcutaneous heparin in addition to aspirin caused more bleeding complications; there was a small reduction in reinfarction and mortality during the treatment period, especially when the results were combined with those of GISSI-2,3,4 but this decrease was not reflected in 35-day or 6-month mortality. Most clinicians will wish to continue with a selective policy of subcutaneous heparin if there is a definite risk of deep vein thrombosis or left ventricular
aneurysm.5,6 As the researchers are careful to point out, the possibility that anistreplase or duteplase might perform better than streptokinase in different circumstances or when combined in different regimens is not excluded. Anistreplase was associated with a slightly greater risk of allergic reactions and
haemorrhage than streptokinase, but these sideeffects might easily be outweighed if simplicity of administration led to earlier initiation of thrombolysis in a greater proportion of eligible patients. Duteplase was associated with an increased risk of haemorrhage, including haemorrhagic stroke, a reduced risk of reinfarction, and no overall mortality difference. In a trial of this size, which is designed to show small differences, statistical significance does not necessarily indicate clinical significance-the excess risk of stroke
EDITORIALS
Enzymes as autoantigens Autoimmunity
accounts tor about 80% ot
newly
Addison’s disease.’ This type of adrenal failure can be associated with two distinct clusters of
diagnosed
disorders.2Type 1 polyendocrine autoimmunity and is usually recognised in the coincidence of chronic by mucocutaneous candidosis and hypoparathyroidism; Addison’s disease ensues 5-10 years later. The much commoner type 2 syndrome is the association of at least two of the following conditions: Addison’s disease, autoimmune hypothyroidism, Graves’ disease, type 1 diabetes, and premature ovarian failure. Both syndromes may be accompanied by other autoimmune conditions--eg, vitiligo and pernicious anaemia-but there are several distinctive features. In particular, the type 1 syndrome has an autosomal recessive inheritance, not linked to genes in the HLA-D region; the type 2 syndrome has a dominant inheritance pattern and strong links with HLA-DR3.3-5 Patients with type 2 syndrome present much later than those with type 1. Immunological heterogeneity is suggested by the presence of steroid cell autoantibodies in over 85% of patients with the type 1 syndrome1,6 whereas these antibodies are found in less than 20 % of patients with sporadic or type-2-associated Addison’s disease.6,7 The high frequency of autoantibodies presumably explains why gonadal failure commonly occurs in type 1 syndrome. From immunofluorescence staining patterns, steroid cell antibodies seem to react with several antigens, which are consistently present in the adrenal but have variable distribution in the ovary, testis, and placenta.8 Adrenal autoantibodies that do not cross-react with antigens in other steroid cells are found in about two-thirds of all Addison’s patients (excluding those with type 1 syndrome).1,9 The nature of these shared and unique autoantigens remains unknown. By contrast, the identity of the thyroid microsomal antigen in thyroiditis’O and of beta cell
syndrome
childhood
is
rare
autoantigens
in type 1 diabetes11,12 has
elucidated. The report
by Krohn and colleagues
lately been
in this issue (p 770) begins to redress that imbalance. Using sera from patients with type 1 polyendocrine autoimmunity to screen an expressed fetal adrenal cDNA library, these researchers obtained and sequenced immunoreactive clones; all but 1 corresponded to part of the known sequence of 17a-hydroxylase. This enzyme is essential for testosterone and oestrogen production, so it is highly likely that 17a-hydroxylase is also one of the gonadal steroid cell autoantigens recognised by autoantibodies. The striking negative finding of this paper (albeit made with only two samples) is the lack of reactivity with 17a-hydroxylase of sera from patients with sporadic Addison’s disease. These sera did not even give an immunoprecipitation reaction with adrenal homogenate, probably because the titre of adrenal antibodies was low. This observation suggests that autoantibodies in patients with sporadic and type-2-associated Addison’s disease recognise adrenal antigens other than 17a-hydroxylase. Patients with the type 1 syndrome may likewise have multiple adrenal autoantibodies, since immunoblotting showed that such sera reacted with four separate proteins whose molecular weights differed from that of 17a-hydroxylase. However, further work is needed to exclude the possibility that these proteins were proteolytic fragments of the enzyme. As Krohn et al point out, the 20-week fetal adrenal used to prepare their cDNA library may have been too immature to express the complete adult repertoire of proteins, and other autoantigenic steroid enzymes are now being
sought. Characterisation of the autoantigens has given a fresh impetus to research into the pathogenesis of adrenal failure in Addison’s disease. One possibility is that autoantibodies may impair target organ enzyme function, but this general mechanism remains equivocal even for the best characterised enzyme autoantigen, thyroid peroxidase. 13,11 It is unclear how readily antibodies have access to intracellular enzymes such as 17a-hydroxylase in vivo or how adrenal cell surface autoantigens identified by immunofluorescence are related to those in the cytoplasm.1s Similar difficulties with access may likewise reduce the pathogenic importance of steroid enzyme complement fixation by autoantibodies. If complement-mediated adrenal injury does occur, it could operate through more subtle mechanisms than simple cell lysis-target cells may respond to sublethal complement attack with reduced metabolic responses and release of inflammatory mediators.16 Peripheral blood T-cell responses to thyroid peroxidase and glutamic acid decarboxylase peptides have been described in Hashimoto’s thyroiditis and type 1 diabetes, respectively. 11-19 These findings raise the alternative possibility that production of autoantibodies against
780
enzymes is
secondary phenomenon, the initiating event being T-cell mediated cytotoxicity directed against endogenous, enzyme-derived peptides coexpressed with class I (and even class II) major histocompatibility complex molecules on target cells. This report is the latest in a series showing that previously ill-defined autoantigens turn out to be enzymes. To the list provided by Krohn et al we can add carboxypeptidase-H in diabetes,12 and myeloperoxidase and a 29 kDa neutrophil proteinase in systemic vasculitis.2O,21 These observations should not come as a surprise: we know that many proteins function as enzymes (at least 10 000 in man) and that the distribution of a proportion of these molecules is restricted. Clearly target-organ-specific enzymes should be considered as candidate autoantigens in a
other obscure autoimmune disorders. 1. Irvine
WJ, Barnes EW. Addison’s disease, ovarian failure and
hypoparathyroidism. Clin Endocrinol Metab 1975; 4: 379-434. 2. Neufeld JDN, Maclaren NK, Blizzard RM. Two types of autoimmune Addison’s disease associated with different polyglandular autoimmune (PGA) syndromes. Medicine 1981; 60: 355-62. 3. Ahonen P, Koskimies S, Likki M-L, Tillikainen A, Perheentupa J. The expression of autoimmune polyglandular disease type 1 appears associated with several HLA-A antigens but not with HLA-DR. J Clin Endocrinol Metab 1988; 66: 1151-57. 4. Eisenbarth GS, Jackson RA. Immunogenetics of polyglandular failure and related diseases. In: Farid NR, ed. HLA in endocrine and metabolic disorders. New York: Academic Press, 1981: 235-64. 5. Maclaren NK, Riley WJ. Inherited susceptibility to autoimmune Addison’s disease is linked to human leukocyte antigens-DR3 and/or DR4, except when associated with type 1 autoimmune polyglandular syndrome. J Clin Endocrinol Metab 1986; 62: 455-59. 6. Ahonen P, Miettinen A, Perheentupa J. Adrenal and steroidal cell antibodies in patients with autoimmune polyglandular disease type 1 and risk of adrenocortical and ovarian failure. J Clin Endocrinol Metab 1987; 64: 494-500. 7. Elder M, Maclaren N, Riley W. Gonadal autoantibodies in patients with hypogonadism and/or Addison’s disease. J Clin Endocrinol Metab 1981; 52: 1137-42. 8. Sotsiou F, Bottazzo GF, Doniach D. Immunofluorescence studies on autoantibodies to steroid-producing cells, and to germline cells in endocrine diseases and infertility. Clin Exp Immunol 1980; 39: 97-111. 9. Latinne D, Vandeput Y, De Bruyere M, Bottazzo F, Sokal G, Crabbe J. Addison’s disease: immunological aspects. Tissue Antigens 1987; 30: 23-24. 10. Czarnocka B, Ruf J, Ferrand M, Carayon P, Lissitky S. Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases. FEBS Lett 1985; 190: 147-51. 11. Baekkeskov S, Jan-Aanstoot H, Christgau S, et al. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABAsynthesizing enzyme glutamine acid decarboxylase. Nature 1990; 347: 151-56. 12. Castano L, Russo E, Zhou L, Lipes MA, Eisenbarth GS. Identification and cloning of a granule autoantigen (carboxypeptidase-H) associated with type 1 diabetes. J Clin Endocrinol Metab 1991; 73: 1197-201. 13. Kohno Y, Yamaguchi F, Saito K, Niimi H, Nishikawa T, Hosoya T. Anti-thyroid peroxidase antibodies in sera from healthy subjects and from patients with chronic thyroiditis: differences in the ability to inhibit thyroid peroxidase activities. Clin Exp Immunol 1991; 85: 459-63. 14. Saller B, Hormann R, Mann K. Heterogeneity of autoantibodies against thyroid peroxidase in autoimmune thyroid disease: evidence against antibodies directly inhibiting peroxidase activity as regulatory factors in thyroid hormone metabolism. J Clin Endocrinol Metab 1991; 72: 188-95. 15. Khoury EL, Hammond L, Bottazzo GF, Doniach D. Surface-reactive antibodies to human adrenal cells in Addison’s disease. Clin Exp Immunol 1981; 45: 48-55. 16. Morgan BP. Complement membrane attack on nucleated cells: resistance, recovery and non-lethal effects. Biochem J 1989; 264: 1-14. 17. Dayan CM Londei M, Corcoran AE, et al. Autoantigen recognition by thyroid-infiltrating T cells in Graves’ disease. Proc Natl Acad Sci USA 1991; 88: 7415-19.
18. Tandon N, Freeman M, Weetman AP. T cell responses to synthetic thyroid peroxidase peptides in autoimmune thyroid disease. Clin Exp Immunol 1991; 86: 56-60. 19. Atkinson MA, Kaufman DL, Campbell L, et al. Response of peripheral-blood mononuclear cells to glutamate decarboxylase in insulin-dependent diabetes. Lancet 1992; 339: 458-59. 20. Falk RJ, Jennett JC. Anti-neutrophil cytoplasmic autoantibodies with specificity for myeloperoxidase in patients with systemic vasculitis and idiopathic necrotizing and crescentic glomerulonephritis. N EnglJ Med 1988; 318: 1651-57. 21. Ludemann J, Utecht B, Gross WL. Anti-neutrophil cytoplasm antibodies in Wegener’s granulomatosis recognize an elastinolytic enzyme. J Exp Med 1990; 171: 357-62.
Streptokinase plus aspirin does the trick: ISIS-3 The Second International Study of Infarct Survival (ISIS-2) confirmed the benefits of streptokinase thrombolysis in acute myocardial infarction shown in the Italian GISSI-1 trial and also showed enhanced survival when streptokinase was combined with aspirin-thrombolysis quickly became established as standard therapy for heart attacks.1,2 ISIS-3, reported on p 753, had the more difficult task of investigating, first, whether the addition of high-dose subcutaneous heparin to aspirin conferred any additional benefit and whether recombinant second, tissue-type activator or plasminogen (duteplase) anisoylated plasminogen streptokinase activator complex (anistreplase) was any better than streptokinase. The clearest message to emerge from this massive enterprise (41 299 patients randomised to different thrombolytic regimens) is that both null hypotheses survived intact-ie, no thrombolytic regimen performed better, in terms of 35-day survival, than the simple and cheap ISIS-2 cocktail. Use of subcutaneous heparin in addition to aspirin caused more bleeding complications; there was a small reduction in reinfarction and mortality during the treatment period, especially when the results were combined with those of GISSI-2,3,4 but this decrease was not reflected in 35-day or 6-month mortality. Most clinicians will wish to continue with a selective policy of subcutaneous heparin if there is a definite risk of deep vein thrombosis or left ventricular
aneurysm.5,6 As the researchers are careful to point out, the possibility that anistreplase or duteplase might perform better than streptokinase in different circumstances or when combined in different regimens is not excluded. Anistreplase was associated with a slightly greater risk of allergic reactions and
haemorrhage than streptokinase, but these sideeffects might easily be outweighed if simplicity of administration led to earlier initiation of thrombolysis in a greater proportion of eligible patients. Duteplase was associated with an increased risk of haemorrhage, including haemorrhagic stroke, a reduced risk of reinfarction, and no overall mortality difference. In a trial of this size, which is designed to show small differences, statistical significance does not necessarily indicate clinical significance-the excess risk of stroke