ADONIS
Clin. exp. Immunol. (1991) 83, 267-273
0009910491000495
Detection of human and murine common idiotypes of DNA antibodies in tissues and sera of patients with autoimmune diseases R. A. WATTS, C. T. RAVIRAJAN*, L. S. WILKINSON, W. WILLIAMS, M. GRIFFITHSt, D. BUTCHERt, A. T. HORSFALL$, N. A. STAINES* & D. A. ISENBERG The Bloomsbury Rheumatology Unit, London, *Immunology Section, King's College, London, tDepartment of Rheumatology Research and Department of Histopathology, University College and Middlesex School of Medicine, London, and t Matilda and Terence Kennedy Institute of Rheumatology, London, England
(Acceptedfor publication 13 September 1990)
SUMMARY The expression in tissue and serum of a panel of murine and human common DNA antibody idiotypes (Ids) (BEG 2, PR 4, F-423, 1-402, II-28, IV-228, V-88) has been investigated. The murine V-88 Id was detected in eight out of 10 and the human BEG 2 Id in five out of 10 labial biopsies from patients with Sjogren's syndrome. The murine F-423, 1-402 and IV-228 Ids were identified in one out of 10 biopsies. In each case the pattern of staining was similar with staining of the acinar basement membrane and a cell population. Using double-labelling immunohistochemistry this cell population were identified as plasma cells. No staining was seen in four normal labial biopsies. The V-88 Id was detected on the epithelial aspect of the thickened basement membrane in three out of nine renal biopsies from patients with systemic lupus erythematosus (SLE). None of the other Ids (BEG 2, PR4, IV-228, F-423 or 1-402) could be detected in renal tissue. None of the Ids were found in skin biopsies from SLE patients. Id V-88 may, like the 16/6 Id to which it is phenotypically related, play a role in the pathogenesis of renal lesions in SLE. The BEG 2 Id could be detected in the serum of patients with rheumatoid arthritis (RA) and active untreated tuberculosis. Ids 11-28, V-88 and 1-402 were elevated in serum from patients with Sj6gren's syndrome and II-28 Id in serum from patients with myositis and RA. None of the Ids were elevated in serum from patients with SLE. Apart from the BEG 2 Id, none of the Ids were elevated in serum from patients with tuberculosis or Gram-negative infections. The presence of murine Ids in human tissue and serum suggests that they are cross-species idiotypes and have been conserved through evolution. Keywords DNA antibody idiotypes systemic
lupus
erythematosus Sjogren's syndrome
globulin eluted from the glomeruli of these patients is enriched in antibody subsets known to be associated with nephritis and it was suggested that Ids GNI and GN2 may be markers of antibodies with nephritogenic properties (Kalunian et al., 1989). Primary Sjdgren's syndrome is characterized by a lymphocytic infiltration of the salivary glands. It has been proposed that autoantibodies are produced locally in the glands and that their production may be under anti-idiotypic control (Horsfall et al., 1988a, 1988b). Private idiotypes on anti-La/SS-B antibodies are located on immunoglobulins deposited in the salivary gland as peri-acinar deposits and on the surface of plasma cells (Horsfall et al., 1988b). Rheumatoid factor idiotypes (for example 17-109) are present in the cytoplasm of up to 5-5% of salivary gland lymphocytes (Fox et al., 1986). The inflammatory infiltrate has a predominance of CD4+ T cells over CD8+ T cells (Fox, Carstens & Fong, 1982; Isenberg et al., 1984b) and it was suggested that this may contribute to the persistent production of anti-La antibodies which in turn can stimulate the secondary production of rheumatoid factors (Horsfall et al., 1988b).
INTRODUCTION Antibodies against both ssDNA and dsDNA are believed to contribute to the renal and skin lesions of systemic lupus erythematosus (SLE) (Landry & Sam, 1973; Brentjens et al., 1977). Furthermore, the common human DNA antibody idiotypes (Ids) 16/6 and 32/15 have been shown to be deposited on immunoglobulins located in the glomerular basement membrane, mesangial cell cytoplasm and focal tuft proliferations of kidneys from patients with SLE (Isenberg & Collins, 1985) and at the dermo-epidermal junction in their skin (Isenberg et al., 1985). Recently, the GNl and GN2 idiotypes originally described on anti-DNA antibodies eluted from the kidneys of NZB/W mice have been shown to be present in the glomeruli of patients with lupus nephritis. Idiotype GN2-positive immunoCorrespondence: Dr R. A. Watts, Rheumatology Research Unit, Unit E6, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK.
267
R. A. Watts et al.
268
Here we describe the distribution of two human and five murine common DNA idiotypes in the serum and tissues (kidney, skin and salivary glands) of patients with SLE and Sjdgren's syndrome. In addition, we report the presence of these idiotypes in the serum, from patients with other autoimmune rheumatic conditions and infectious diseases. MATERIALS AND METHODS Human sera Coded sera were obtained from 75 patients with SLE (including six bled serially on four occasions during periods of active and inactive disease); 25 patients with rheumatoid arthritis (RA); 25 healthy first-degree relatives of SLE patients; 20 patients with primary Sj6gren's syndrome; 20 patients with myositis; 15 patients with autoimmune liver disease (chronic active hepatitis or primary biliary cirrhosis); and 15 patients with scleroderma. In addition, sera from the spouses of 15 SLE patients (10 included in the study); 25 with active untreated tuberculosis; 13 with Klebsiella infections, 13 with Escherichia coli infections and 17 patients with ankylosing spondylitis were also included. As healthy controls sera were obtained from up to 29 healthy individuals who had no personal or family history of autoimmune diseases. Each of the SLE patients met four or more of the American Rheumatism Association (ARA) revised criteria for the classification of the disease (Tan et al., 1982). Disease activity was graded according to a previously published index (Isenberg, Shoenfeld & Schwartz, 1984a), which has recently been validated with an activity index generated by a more detailed computer-based programme (Symmonds et al., 1988). This index was used to designate the patients as inactive (grade 1), mildly active (grade 2), moderately active (grade 3), or severely active (grade 4). The patients with RA each met four or more of the revised ARA criteria (Arnett et al., 1988). Those with primary Sjdgren's syndrome were diagnosed by the criteria of Isenberg et al. (1984b). The patients with adult-onset myositis had three or four of the criteria of Bohan & Peter (1975). The scleroderma patients all met the preliminary criteria of the ARA (Masi, Rodnan & Medsger, 1981) and the patients with autoimmune liver disease had all been diagnosed histologically. The patients with the infectious diseases were diagnosed according to standard laboratory techniques, the patients with ankylosing spondylitis were diagnosed according to the New York criteria (Bennett & Burch, 1966).
Monoclonal antibodies (MoAbs) and anti-idiotypes The production, purification and specificity of monoclonal antibody BEG 2 has been described (Watts et al., 1990). Hepatic lymphocytes from a 12-week-old human fetus were fused with GM4672 cells. Monoclonal antibody BEG 2 binds preferentially to ssDNA but also to dsDNA, poly deoxythymidilic acid, polyinosinic acid and poly (ADP-ribose) but not to cardiolipin or RNA. The derivation of MoAb PR4 from the peripJheral blood lymphocytes of a patient with leprosy and its binding specificity has been described (Williams et al., 1988). The preparation, purification and binding specificities of the murine MoAbs F-423, 1-402, IV-228, 11-28 and V-88 have been described (Morgan et al., 1985a). F-423 was derived from a 15day-old fetal MRL/Mp-lpr/lpr (MRL/lpr) mouse, 11-28 and V-
88 were derived from adult female (NZB/W) Fl mice, and 1-402 and IV-228 were derived from adult male MRL/lpr mice. All the murine MoAbs bind to ssDNA and MoAbs 11-28, V-88, F-423 and 1-402 bind to dsDNA. Polyclonal anti-idiotype sera were raised by immunization of rabbits with MoAbs BEG 2 and PR 4. Anti-idiotype antibodies were purified by affinity chromatography using a human immunoglobulin Sepharose 4B column (Pharmacia, Milton Keynes, UK) followed by a homologous MoAb Sepharose column (Williams et al., 1988; Watts et al., 1990). Antiidiotypic sera to murine MoAbs were raised in a comparable manner. ELISA for detection ofBEG 2 Id and PR4 Id on immunoglobulin in human sera Capture ELISAs which have been described were used to detect the BEG 2 Id and PR 4 Id in human sera (Williams et al., 1988; Watts et al., 1990). The upper limit of normal for the BEG 2 assay was set arbitrarily at the 90th centile of the OD values of the 29 normal healthy control sera expressed as a percentage ofa standard reference serum. The upper limit of normal for PR4 Id was set as the mean+2 s.d. of values obtained for the normal control sera (Williams et al., 1988). ELISA for detection murine Ids on immunoglobulin in human serum These were detected as described using a capture ELISA (Isenberg et al., 1990). A panel of normal sera was used to establish the normal range and the upper limit of normal (mean+ 2 s.d.).
ELISA for quantifying immunoglobulin Serum IgG and IgM were measured using a previously described capture ELISA (Watts et al., 1989). ELISA for detection of DNA binding activity Serum DNA binding activity was measured using a previously described direct binding ELISA (Watts et al., 1990). Detection of idiotypes in human tissue Tissue sections. Frozen skin biopsy sections were prepared from 10 patients with SLE and 12 non-SLE patients with immunoglobulin deposits in the skin. Frozen labial biopsy specimens from nine patients with secondary Sj6gren's syndrome, one with primary Sjogren's syndrome and four normal subjects were obtained. Sera were obtained simultaneously from eight of these patients. Paraffin sections of renal biopsy material from nine patients with SLE and seven without SLE but known to have IgG deposition were obtained. Frozen sections of 6-pm thickness were cut on a SLEE TE microtome (Histological Equipment, Carshalton, UK) and taken up on to glass slides. Sections were dried for 30 min under a cool fan and then in open containers overnight at room temperature. Sections were stored wrapped in foil in hermetically sealed boxes at - 70°C until required. Prior to staining, sections were warmed to room temperature before unwrapping, and then immersed for 10 min in cold acetone. The paraffin sections were de-waxed using Xylene followed by three washes of 5 min each in absolute ethanol and one each of 70% and 50% ethanol before water.
Presence of DNA antibody idiotypes in human tissue and serum
269
Table 1. Percentage raised levels of human and murine idiotypes in human sera
BEG 2 PR4 11-28 IV-228 V-88 1-402 F-423
Normals SLE Sj6gren's syndrome Myositis RA Scleroderma Liver disease SLE relatives SLE spouses TB infection Klebsiella infection Escherichia coli infection Ankylosing spondylitis
7 9 0 0 23 13 0 5 0 34 14 15 0
7 9 0 10 52* 0
40t 0 0 NT NT NT NT
8 10 58* 47* 52* 0 14 40
8 6 21 0 4 7 0 12
0 0 8 8 0
8 17 5 32 7 7 16
8 6 68* 5 8 7 0 24
8 7 16 0 28 0 0 4
0 0 0 8
0 13 8 8
0 8 0 0
0 8 15 8
6
0
0
0
47t
SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; TB, tuberculosis; NT, not tested. * P
Clin. exp. Immunol. (1991) 83, 267-273
0009910491000495
Detection of human and murine common idiotypes of DNA antibodies in tissues and sera of patients with autoimmune diseases R. A. WATTS, C. T. RAVIRAJAN*, L. S. WILKINSON, W. WILLIAMS, M. GRIFFITHSt, D. BUTCHERt, A. T. HORSFALL$, N. A. STAINES* & D. A. ISENBERG The Bloomsbury Rheumatology Unit, London, *Immunology Section, King's College, London, tDepartment of Rheumatology Research and Department of Histopathology, University College and Middlesex School of Medicine, London, and t Matilda and Terence Kennedy Institute of Rheumatology, London, England
(Acceptedfor publication 13 September 1990)
SUMMARY The expression in tissue and serum of a panel of murine and human common DNA antibody idiotypes (Ids) (BEG 2, PR 4, F-423, 1-402, II-28, IV-228, V-88) has been investigated. The murine V-88 Id was detected in eight out of 10 and the human BEG 2 Id in five out of 10 labial biopsies from patients with Sjogren's syndrome. The murine F-423, 1-402 and IV-228 Ids were identified in one out of 10 biopsies. In each case the pattern of staining was similar with staining of the acinar basement membrane and a cell population. Using double-labelling immunohistochemistry this cell population were identified as plasma cells. No staining was seen in four normal labial biopsies. The V-88 Id was detected on the epithelial aspect of the thickened basement membrane in three out of nine renal biopsies from patients with systemic lupus erythematosus (SLE). None of the other Ids (BEG 2, PR4, IV-228, F-423 or 1-402) could be detected in renal tissue. None of the Ids were found in skin biopsies from SLE patients. Id V-88 may, like the 16/6 Id to which it is phenotypically related, play a role in the pathogenesis of renal lesions in SLE. The BEG 2 Id could be detected in the serum of patients with rheumatoid arthritis (RA) and active untreated tuberculosis. Ids 11-28, V-88 and 1-402 were elevated in serum from patients with Sj6gren's syndrome and II-28 Id in serum from patients with myositis and RA. None of the Ids were elevated in serum from patients with SLE. Apart from the BEG 2 Id, none of the Ids were elevated in serum from patients with tuberculosis or Gram-negative infections. The presence of murine Ids in human tissue and serum suggests that they are cross-species idiotypes and have been conserved through evolution. Keywords DNA antibody idiotypes systemic
lupus
erythematosus Sjogren's syndrome
globulin eluted from the glomeruli of these patients is enriched in antibody subsets known to be associated with nephritis and it was suggested that Ids GNI and GN2 may be markers of antibodies with nephritogenic properties (Kalunian et al., 1989). Primary Sjdgren's syndrome is characterized by a lymphocytic infiltration of the salivary glands. It has been proposed that autoantibodies are produced locally in the glands and that their production may be under anti-idiotypic control (Horsfall et al., 1988a, 1988b). Private idiotypes on anti-La/SS-B antibodies are located on immunoglobulins deposited in the salivary gland as peri-acinar deposits and on the surface of plasma cells (Horsfall et al., 1988b). Rheumatoid factor idiotypes (for example 17-109) are present in the cytoplasm of up to 5-5% of salivary gland lymphocytes (Fox et al., 1986). The inflammatory infiltrate has a predominance of CD4+ T cells over CD8+ T cells (Fox, Carstens & Fong, 1982; Isenberg et al., 1984b) and it was suggested that this may contribute to the persistent production of anti-La antibodies which in turn can stimulate the secondary production of rheumatoid factors (Horsfall et al., 1988b).
INTRODUCTION Antibodies against both ssDNA and dsDNA are believed to contribute to the renal and skin lesions of systemic lupus erythematosus (SLE) (Landry & Sam, 1973; Brentjens et al., 1977). Furthermore, the common human DNA antibody idiotypes (Ids) 16/6 and 32/15 have been shown to be deposited on immunoglobulins located in the glomerular basement membrane, mesangial cell cytoplasm and focal tuft proliferations of kidneys from patients with SLE (Isenberg & Collins, 1985) and at the dermo-epidermal junction in their skin (Isenberg et al., 1985). Recently, the GNl and GN2 idiotypes originally described on anti-DNA antibodies eluted from the kidneys of NZB/W mice have been shown to be present in the glomeruli of patients with lupus nephritis. Idiotype GN2-positive immunoCorrespondence: Dr R. A. Watts, Rheumatology Research Unit, Unit E6, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK.
267
R. A. Watts et al.
268
Here we describe the distribution of two human and five murine common DNA idiotypes in the serum and tissues (kidney, skin and salivary glands) of patients with SLE and Sjdgren's syndrome. In addition, we report the presence of these idiotypes in the serum, from patients with other autoimmune rheumatic conditions and infectious diseases. MATERIALS AND METHODS Human sera Coded sera were obtained from 75 patients with SLE (including six bled serially on four occasions during periods of active and inactive disease); 25 patients with rheumatoid arthritis (RA); 25 healthy first-degree relatives of SLE patients; 20 patients with primary Sj6gren's syndrome; 20 patients with myositis; 15 patients with autoimmune liver disease (chronic active hepatitis or primary biliary cirrhosis); and 15 patients with scleroderma. In addition, sera from the spouses of 15 SLE patients (10 included in the study); 25 with active untreated tuberculosis; 13 with Klebsiella infections, 13 with Escherichia coli infections and 17 patients with ankylosing spondylitis were also included. As healthy controls sera were obtained from up to 29 healthy individuals who had no personal or family history of autoimmune diseases. Each of the SLE patients met four or more of the American Rheumatism Association (ARA) revised criteria for the classification of the disease (Tan et al., 1982). Disease activity was graded according to a previously published index (Isenberg, Shoenfeld & Schwartz, 1984a), which has recently been validated with an activity index generated by a more detailed computer-based programme (Symmonds et al., 1988). This index was used to designate the patients as inactive (grade 1), mildly active (grade 2), moderately active (grade 3), or severely active (grade 4). The patients with RA each met four or more of the revised ARA criteria (Arnett et al., 1988). Those with primary Sjdgren's syndrome were diagnosed by the criteria of Isenberg et al. (1984b). The patients with adult-onset myositis had three or four of the criteria of Bohan & Peter (1975). The scleroderma patients all met the preliminary criteria of the ARA (Masi, Rodnan & Medsger, 1981) and the patients with autoimmune liver disease had all been diagnosed histologically. The patients with the infectious diseases were diagnosed according to standard laboratory techniques, the patients with ankylosing spondylitis were diagnosed according to the New York criteria (Bennett & Burch, 1966).
Monoclonal antibodies (MoAbs) and anti-idiotypes The production, purification and specificity of monoclonal antibody BEG 2 has been described (Watts et al., 1990). Hepatic lymphocytes from a 12-week-old human fetus were fused with GM4672 cells. Monoclonal antibody BEG 2 binds preferentially to ssDNA but also to dsDNA, poly deoxythymidilic acid, polyinosinic acid and poly (ADP-ribose) but not to cardiolipin or RNA. The derivation of MoAb PR4 from the peripJheral blood lymphocytes of a patient with leprosy and its binding specificity has been described (Williams et al., 1988). The preparation, purification and binding specificities of the murine MoAbs F-423, 1-402, IV-228, 11-28 and V-88 have been described (Morgan et al., 1985a). F-423 was derived from a 15day-old fetal MRL/Mp-lpr/lpr (MRL/lpr) mouse, 11-28 and V-
88 were derived from adult female (NZB/W) Fl mice, and 1-402 and IV-228 were derived from adult male MRL/lpr mice. All the murine MoAbs bind to ssDNA and MoAbs 11-28, V-88, F-423 and 1-402 bind to dsDNA. Polyclonal anti-idiotype sera were raised by immunization of rabbits with MoAbs BEG 2 and PR 4. Anti-idiotype antibodies were purified by affinity chromatography using a human immunoglobulin Sepharose 4B column (Pharmacia, Milton Keynes, UK) followed by a homologous MoAb Sepharose column (Williams et al., 1988; Watts et al., 1990). Antiidiotypic sera to murine MoAbs were raised in a comparable manner. ELISA for detection ofBEG 2 Id and PR4 Id on immunoglobulin in human sera Capture ELISAs which have been described were used to detect the BEG 2 Id and PR 4 Id in human sera (Williams et al., 1988; Watts et al., 1990). The upper limit of normal for the BEG 2 assay was set arbitrarily at the 90th centile of the OD values of the 29 normal healthy control sera expressed as a percentage ofa standard reference serum. The upper limit of normal for PR4 Id was set as the mean+2 s.d. of values obtained for the normal control sera (Williams et al., 1988). ELISA for detection murine Ids on immunoglobulin in human serum These were detected as described using a capture ELISA (Isenberg et al., 1990). A panel of normal sera was used to establish the normal range and the upper limit of normal (mean+ 2 s.d.).
ELISA for quantifying immunoglobulin Serum IgG and IgM were measured using a previously described capture ELISA (Watts et al., 1989). ELISA for detection of DNA binding activity Serum DNA binding activity was measured using a previously described direct binding ELISA (Watts et al., 1990). Detection of idiotypes in human tissue Tissue sections. Frozen skin biopsy sections were prepared from 10 patients with SLE and 12 non-SLE patients with immunoglobulin deposits in the skin. Frozen labial biopsy specimens from nine patients with secondary Sj6gren's syndrome, one with primary Sjogren's syndrome and four normal subjects were obtained. Sera were obtained simultaneously from eight of these patients. Paraffin sections of renal biopsy material from nine patients with SLE and seven without SLE but known to have IgG deposition were obtained. Frozen sections of 6-pm thickness were cut on a SLEE TE microtome (Histological Equipment, Carshalton, UK) and taken up on to glass slides. Sections were dried for 30 min under a cool fan and then in open containers overnight at room temperature. Sections were stored wrapped in foil in hermetically sealed boxes at - 70°C until required. Prior to staining, sections were warmed to room temperature before unwrapping, and then immersed for 10 min in cold acetone. The paraffin sections were de-waxed using Xylene followed by three washes of 5 min each in absolute ethanol and one each of 70% and 50% ethanol before water.
Presence of DNA antibody idiotypes in human tissue and serum
269
Table 1. Percentage raised levels of human and murine idiotypes in human sera
BEG 2 PR4 11-28 IV-228 V-88 1-402 F-423
Normals SLE Sj6gren's syndrome Myositis RA Scleroderma Liver disease SLE relatives SLE spouses TB infection Klebsiella infection Escherichia coli infection Ankylosing spondylitis
7 9 0 0 23 13 0 5 0 34 14 15 0
7 9 0 10 52* 0
40t 0 0 NT NT NT NT
8 10 58* 47* 52* 0 14 40
8 6 21 0 4 7 0 12
0 0 8 8 0
8 17 5 32 7 7 16
8 6 68* 5 8 7 0 24
8 7 16 0 28 0 0 4
0 0 0 8
0 13 8 8
0 8 0 0
0 8 15 8
6
0
0
0
47t
SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; TB, tuberculosis; NT, not tested. * P
