CGn. exp. Immunol. (1977) 28, 546-547.
BRIEF COMMUNICATION
Reticulin autoantibodies in childhood coeliac disease not directed against type III collagen R. TIMPL, * G. WI CKt & G. GRAND ITS CH * Max-Planck-Institute for Biochemistry, Martinsried, Munich, Germany. t Institute for General and Experimental Pathology, University of Innsbruck, Innsbruck and + University Children's Hospital, Vienna, Austria (Received 1 February 1977)
Circulating autoantibodies to reticulin demonstrable by indirect immunofluorescence (11F) occur with high frequency in patients with coeliac disease (Seah et al., 1971). We have recently shown that rabbit antibodies to type III collagen and procollagen show a strong reaction with reticulin in various tissues while antisera to the other three known types (I, II and IV) do not (Nowack et al., 1976). It was therefore deemed of interest to study whether the autoantibodies to reticulin found in childhood coeliac disease might react with these collagenous components. The sera of thirty-one children of both sexes with coeliac disease were studied: fourteen untreated children were listed under the diagnosis of florid disease (maximally 1 month on a gluten-free diet), seventeen patients were under prolonged diet and found to be in remission. The diagnostic criteria applied were described in detail recently (Granditsch & Wick, 1976). An age- and sex-matched group of fifteen children presenting mainly with diarrhoea and failure to thrive and four apparently healthy children investigated as relatives of coeliac patients were studied for control purposes. Sera of these four groups were screened in II F tests on unfixed, frozen 4-jim thick sections of rat liver for the presence of reticulin autoantibodies (Granditsch & Wick, 1976). Over 70% were positive in both groups of patients with coeliac disease irrespective of their diet. One serum was found to be positive among the nineteen controls. In addition these sera were examined for their reaction with type III collagen and procollagen by means of passive haemagglutination (PHA) tests (Beil, Timpl & Furthmayr, 1973) and radioimmunoassays (RIA) (Adelmann, Gentner & Hopper, 1973). Type III collagen was prepared by limited pepsin digestion from normal human skin (Chung & Miller, 1974). Neutral salt-extracted type III collagen and procollagen derived from foetal bovine skin was also used (Timpl et al., 1975). Previous 11F data have demonstrated a strong antigenic cross-reaction between human and bovine type III collagen and procollagen (Nowack et al., 1976). Immunochemical analysis (Becker et al., 1976) has shown that the major antigenic determinant of type III collagen resides in a short, non-helical segment at the amino terminal of the molecule (peptide TIX). The human sera where therefore also examined for their reaction with peptide TIX in RIA. Sera from both control groups showed only low titres in PHA and little binding of the three antigens (Table 1). With the exception of one serum no stronger reaction was found M ith the sera of the two groups with coeliac disease. This exceptional serum gave 340 0 (TIX) and 53° (type III collagen) antigen binding which is significantly above the average binding capacity observed with all other sera. This serum also showed distinct binding of type I collagen. However, a second serum sample drawn from this patient fell within the normal range of antigen binding. Adelmann (personal communication) also found some sera of patients with florid coeliac disease to give positive reactions with type I and III collagen in RIA. These data indicate that autoantibodies to reticulin found in the sera of patients with coeliac disease are not directed against the collagenous component of reticulin fibres. Previous studies with antibodies to collagen have clearly shown a 10-100-fold higher sensitivity of PHA and RIA as compared to 11F Correspondence: Dr R. Timpl, Max-Planck-Institute for Biochemistry, Martinsried, Munich, Germany.
546
547
Reticulin autoantibodies TABLE 1.
Groups
Florid coeliac diseaset
RIA (per cent binding, mean ± s.d.)t PHA (-log2, mean ± s.d.) Reticulin* auto-Ab/total no. Type III collagen Type III procollagen Type III collagen Peptide TIX 10/14
2-2+ 1-2
2-0+0-8
11-5+10-7
2-8+ 27
13/17
2-3+0-8
1-6+0-6
11-5+3-5
3-0+2-8
1/15
2-5+0-6§
1-9+±07§
14-7+4-7§
3-2+3-1§
0/4
2 5+0 6
1-8+0-5
13-6+2-6
4-0+2-4
(n = 14) Coeliac disease on diet, in remission (n = 17) Other gastrointestinal diseases (n = 15) Healthy controls
(n = 4) PHA = Passive haemagglutination; RIA = radioimmunoassay. * Screened in IIF in dilutions of 1: 10 and 1:20. t 0 05 ml serum samples were incubated overnight with 100 ng collagen or 0-1 ng peptide TIX at 4VC prior to precipitation of immunoglobulins by sheep or goat antibodies against total Ig (Cohn Fraction II). t Maximum duration of diet 1 month. § No statistically significant difference to values from other groups (Student's t-test). PHA = Passive haemagglutination; RIA = radioimmunoassay.
(Nowack et al., 1976; Wick et al., 1976). Thus low sensitivity of the assay systems used in the present study do not seem to be responsible for the lack of reaction with type III collagen. From our results and the previous observations of others (Pras & Glynn, 1973) it can be concluded that type III collagen is not a major autoantigen involved in coeliac disease. REFERENCES ADELMANN B.C., GENTNER, G.J. & HOPPER, K. (1973) A sensitive radioimmunoassay for collagen. 3. Immunol. Methods, 3, 319. BECKER, U., NOWACK, H., GAY, S. & TIMPL, R. (1976) Production and specificity of antibodies against the aminoterminal region in type III collagen. Immunology, 31, 57. BEIL, W., TIMPL, R. & FURTHMAYR, H. (1973) Conformation dependence of antigenic determinants on the collagen molecule. Immunology, 24, 13. CHUNG, E. & MILLER, E.J. (1974) Collagen polymorphism: characterization of molecules with the chain composition [Oil (III)]3 in human tissues. Science, 183, 1200. GRANDITSCH, G. & WICK, G. (1976) Immunologische Studien an Kindern mit Coliakie. Klin. Pidiat. 188, 408. NOWACK, H., GAY, S., WICK, G., BECKER, U. & TIMPL, R. (1976) Preparaton and use in immunohistology of anti-
bodies specific for type I and type III collagen and procollagen. A. Immunol. Methods, 12, 117. PRAS, M. & GLYNN, L.E. (1973) Isolation of a noncollagenous reticulin component and its primary characterization. Brit. J. exp. Path. 54, 449. SEAH, P.P., FRY, L., ROSSITER, M.A., HOFFBRAND, A.V. & HOLBOROW, E.J. (1971) Anti-reticulin antibodies in childhood coeliac disease. Lancet, ii, 681. TIMPL, R., GLANVILLE, R.W., NOWACK, H., WIEDEMANN, H., FIETZEK, P.P. & KUHN, K. (1975) Isolation, chemical and electron microscopical characterization of neutral salt-soluble type III collagen and procollagen from fetal bovine skin. Hoppe-Seyler's Z. Physiol. Chem. 356, 1783. WICK, G., NOWACK, H., HAHN, E., TIMPL, R. & MILLER, E.J. (1976) Visualization of type I and II collagens in tissue sections by immunohistological techniques. A. Immunol. 117, 298.
BRIEF COMMUNICATION
Reticulin autoantibodies in childhood coeliac disease not directed against type III collagen R. TIMPL, * G. WI CKt & G. GRAND ITS CH * Max-Planck-Institute for Biochemistry, Martinsried, Munich, Germany. t Institute for General and Experimental Pathology, University of Innsbruck, Innsbruck and + University Children's Hospital, Vienna, Austria (Received 1 February 1977)
Circulating autoantibodies to reticulin demonstrable by indirect immunofluorescence (11F) occur with high frequency in patients with coeliac disease (Seah et al., 1971). We have recently shown that rabbit antibodies to type III collagen and procollagen show a strong reaction with reticulin in various tissues while antisera to the other three known types (I, II and IV) do not (Nowack et al., 1976). It was therefore deemed of interest to study whether the autoantibodies to reticulin found in childhood coeliac disease might react with these collagenous components. The sera of thirty-one children of both sexes with coeliac disease were studied: fourteen untreated children were listed under the diagnosis of florid disease (maximally 1 month on a gluten-free diet), seventeen patients were under prolonged diet and found to be in remission. The diagnostic criteria applied were described in detail recently (Granditsch & Wick, 1976). An age- and sex-matched group of fifteen children presenting mainly with diarrhoea and failure to thrive and four apparently healthy children investigated as relatives of coeliac patients were studied for control purposes. Sera of these four groups were screened in II F tests on unfixed, frozen 4-jim thick sections of rat liver for the presence of reticulin autoantibodies (Granditsch & Wick, 1976). Over 70% were positive in both groups of patients with coeliac disease irrespective of their diet. One serum was found to be positive among the nineteen controls. In addition these sera were examined for their reaction with type III collagen and procollagen by means of passive haemagglutination (PHA) tests (Beil, Timpl & Furthmayr, 1973) and radioimmunoassays (RIA) (Adelmann, Gentner & Hopper, 1973). Type III collagen was prepared by limited pepsin digestion from normal human skin (Chung & Miller, 1974). Neutral salt-extracted type III collagen and procollagen derived from foetal bovine skin was also used (Timpl et al., 1975). Previous 11F data have demonstrated a strong antigenic cross-reaction between human and bovine type III collagen and procollagen (Nowack et al., 1976). Immunochemical analysis (Becker et al., 1976) has shown that the major antigenic determinant of type III collagen resides in a short, non-helical segment at the amino terminal of the molecule (peptide TIX). The human sera where therefore also examined for their reaction with peptide TIX in RIA. Sera from both control groups showed only low titres in PHA and little binding of the three antigens (Table 1). With the exception of one serum no stronger reaction was found M ith the sera of the two groups with coeliac disease. This exceptional serum gave 340 0 (TIX) and 53° (type III collagen) antigen binding which is significantly above the average binding capacity observed with all other sera. This serum also showed distinct binding of type I collagen. However, a second serum sample drawn from this patient fell within the normal range of antigen binding. Adelmann (personal communication) also found some sera of patients with florid coeliac disease to give positive reactions with type I and III collagen in RIA. These data indicate that autoantibodies to reticulin found in the sera of patients with coeliac disease are not directed against the collagenous component of reticulin fibres. Previous studies with antibodies to collagen have clearly shown a 10-100-fold higher sensitivity of PHA and RIA as compared to 11F Correspondence: Dr R. Timpl, Max-Planck-Institute for Biochemistry, Martinsried, Munich, Germany.
546
547
Reticulin autoantibodies TABLE 1.
Groups
Florid coeliac diseaset
RIA (per cent binding, mean ± s.d.)t PHA (-log2, mean ± s.d.) Reticulin* auto-Ab/total no. Type III collagen Type III procollagen Type III collagen Peptide TIX 10/14
2-2+ 1-2
2-0+0-8
11-5+10-7
2-8+ 27
13/17
2-3+0-8
1-6+0-6
11-5+3-5
3-0+2-8
1/15
2-5+0-6§
1-9+±07§
14-7+4-7§
3-2+3-1§
0/4
2 5+0 6
1-8+0-5
13-6+2-6
4-0+2-4
(n = 14) Coeliac disease on diet, in remission (n = 17) Other gastrointestinal diseases (n = 15) Healthy controls
(n = 4) PHA = Passive haemagglutination; RIA = radioimmunoassay. * Screened in IIF in dilutions of 1: 10 and 1:20. t 0 05 ml serum samples were incubated overnight with 100 ng collagen or 0-1 ng peptide TIX at 4VC prior to precipitation of immunoglobulins by sheep or goat antibodies against total Ig (Cohn Fraction II). t Maximum duration of diet 1 month. § No statistically significant difference to values from other groups (Student's t-test). PHA = Passive haemagglutination; RIA = radioimmunoassay.
(Nowack et al., 1976; Wick et al., 1976). Thus low sensitivity of the assay systems used in the present study do not seem to be responsible for the lack of reaction with type III collagen. From our results and the previous observations of others (Pras & Glynn, 1973) it can be concluded that type III collagen is not a major autoantigen involved in coeliac disease. REFERENCES ADELMANN B.C., GENTNER, G.J. & HOPPER, K. (1973) A sensitive radioimmunoassay for collagen. 3. Immunol. Methods, 3, 319. BECKER, U., NOWACK, H., GAY, S. & TIMPL, R. (1976) Production and specificity of antibodies against the aminoterminal region in type III collagen. Immunology, 31, 57. BEIL, W., TIMPL, R. & FURTHMAYR, H. (1973) Conformation dependence of antigenic determinants on the collagen molecule. Immunology, 24, 13. CHUNG, E. & MILLER, E.J. (1974) Collagen polymorphism: characterization of molecules with the chain composition [Oil (III)]3 in human tissues. Science, 183, 1200. GRANDITSCH, G. & WICK, G. (1976) Immunologische Studien an Kindern mit Coliakie. Klin. Pidiat. 188, 408. NOWACK, H., GAY, S., WICK, G., BECKER, U. & TIMPL, R. (1976) Preparaton and use in immunohistology of anti-
bodies specific for type I and type III collagen and procollagen. A. Immunol. Methods, 12, 117. PRAS, M. & GLYNN, L.E. (1973) Isolation of a noncollagenous reticulin component and its primary characterization. Brit. J. exp. Path. 54, 449. SEAH, P.P., FRY, L., ROSSITER, M.A., HOFFBRAND, A.V. & HOLBOROW, E.J. (1971) Anti-reticulin antibodies in childhood coeliac disease. Lancet, ii, 681. TIMPL, R., GLANVILLE, R.W., NOWACK, H., WIEDEMANN, H., FIETZEK, P.P. & KUHN, K. (1975) Isolation, chemical and electron microscopical characterization of neutral salt-soluble type III collagen and procollagen from fetal bovine skin. Hoppe-Seyler's Z. Physiol. Chem. 356, 1783. WICK, G., NOWACK, H., HAHN, E., TIMPL, R. & MILLER, E.J. (1976) Visualization of type I and II collagens in tissue sections by immunohistological techniques. A. Immunol. 117, 298.
