Clin. exp. Immunol. (1992) 87, 438-443
Antibodies against sulphatide in sera from patients with autoimmune rheumatic diseases S. AOTSUKA, M. OKAWA-TAKATSUJI, S. UWATOKO, R. YOKOHARI*, Y. IKEDAt & G. TODAt Division of Immunology, Clinical Research Institute, National Medical Center, Tokyo, *National Atami Hospital, Shizuoka, and tFirst Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan
(Acceptedfor publication 3 October 1991)
SUMMARY We tested sera of patients with various autoimmune rheumatic diseases for the presence of antibodies against sulphatide (an acidic glycosphingolipid), identified as a target antigen for antibodies against the liver cell membrane. Thirty-five percent (7/20) of patients with lupus in the active stage possessed anti-sulphatide antibodies, whereas 10% (2/20) of those in the inactive stage and 20% (4/20) of those in the stationary stage possessed such antibodies. Moreover, 10% (3/29) of patients with other autoimmune rheumatic diseases also possessed anti-sulphatide antibodies. The level of antisulphatide antibodies was significantly correlated with the levels of anti-double-stranded (ds) DNA antibodies (r = 0-634, P < 0-001) and dextran sulphate-binding IgG (r = 0 407, P < 0 001). The serum levels of antibodies against sulphatide were correlated with a history of seizures or psychosis in patients with autoimmune rheumatic diseases. Gels coupled with polyanionic dextran sulphate, monoanionic sulphanilic acid and DNA were shown effectively to adsorb anti-sulphatide antibodies in the sera of patients with active systemic lupus erythematosus (SLE) and autoimmune chronic active hepatitis (Al-CAH). These results suggest that the observed reactivity with sulphatide is due to the presence of antibodies capable of reacting with various anionic molecules in the sera of patients with autoimmune rheumatic diseases as well as those with AI-CAH. Keywords sulphatide anti-dsDNA antibodies heparan sulphate dextran sulphate
INTRODUCTION Anti-DNA antibodies, which serve as significant diagnostic and prognostic markers in systemic lupus erythematosus (SLE), have been shown to have the capacity to react with a wide variety of molecules with repeating negative charges such as cardiolipin and other negatively charged phospholipids [1,2], chondriotin sulphate, heparan sulphate and Cibacron blue F3GA [3,4]. Autoimmune chronic active hepatitis (AI-CAH) is characterized by the presence of autoantibodies that react with various substances including nuclear constituents, smooth muscle cells, and hepatocyte plasma membranes. Recently, it was reported [5] that sera from patients with AT-CAH contain an IgG-class antibody against the acidic glycosphingolipid fraction of the rabbit hepatocyte plasma membranes. Antibodies to sulphatide were frequently found in sera from patients with AT-CAH but rarely in patients with virus-induced chronic active liver disease. The fact that sulphatide contains anionic sulphated residues prompted us to evaluate the antibody specificity and clinical
correlations of anti-sulphatide antibodies in autoimmune rheumatic diseases. PATIENTS AND METHODS Sera Sera from patients with active or inactive SLE (40 paired sera), stationary SLE (20 sera), other connective tissue diseases (29 serum samples: systemic sclerosis (n = 16); rheumatoid arthritis (n=4); mixed connective tissue disease (n=4); polymyositis (n = 3); Sjogren's syndrome (n = 2)), and autoimmune chronic active hepatitis (Al-CAH, 16 sera) were collected, stored at -80 C and thawed at 37°C before use. Al-CAH was diagnosed as described previously [6]. The clinical assessment of SLE disease activity was based on objective clinical disease manifestations and laboratory evidence of disease activity: (i) fever; (ii) arthralgia; (iii) cutaneous erythema; (iv) oral ulcer or alopecia; (v) elevated erythrocyte sedimentation rate (> 30 mm/h); (vi) hypocomplementaemia (CH50 < 20 U/ml or C3 < 60 mg/dl); (vii) leukopenia ( < 4 0 x 109/l); (viii) hypoalbuminaemia ( < 35 g/l); and (ix) positive LE cell or LE test. The activity of SLE was judged at the times of serum sampling. An arbitrary clinical assessment system for SLE disease activity yielded values
Correspondence: Shinichi Aotsuka, Clinical Research Institute, National Medical Center, 1-21-1, Toyama, Shinjuku-ku, Tokyo 162, Japan.
438
Anti-sulphatide antibody in autoimmune rheumatic diseases ranging from over four for active SLE to less than three for inactive or stationary SLE. Stationary SLE was defined as the absence of active disease, at least during our observation period. Based on previous experience, serum enzyme elevations were defined arbitrarily as follows: SGOT > 55 U/i (normal laboratory range 0-45) provided that myositis and haemolysis were absent, and/or SGPT >45 U/i (0-35), and/or ALP > 150 U/I (30-135) provided that the hepatic isoenzyme was demonstrated to be elevated. Patients were evaluated as to whether they had symptoms such as seizures or psychosis in the absence of offending drugs or known metabolic derangements.
Ligands Sorbent gels were prepared by coupling various molecules with different charges to cellulose gel for use as ligands. Dextran sulphate (DXS), purchased from Sigma Chemical Co., St Louis, MO, was immobilized via a hydroxyl group, and other ligands via their amino groups or the thiol group, according to the modified methods described by Vretblad [7]. Another anionic polymer (chondroitin sulphate, alginate), anionic monomers (pyromellitic acid, 2-aminoethyl hydrogen sulphate, sulphanilic acid, taurine), a cationic monomer (lysine), and a hydrophobic monomer (benzyl amine) were also coupled separately to the cellulose gel. DNA cellulose was purchased from Sigma. Anti-sulphatide antibody assay Fifty microlitres of sulphatide in ethanol (10 pg/ml) were placed in the wells of a microtitre plate (Nunc-Immunoplate I-96F; Nunc, Roskilde, Denmark) and evaporated overnight at 37 C. The wells were then washed six times with 0 135 M NaCl/0-0 1 5 M Tris-HCl (pH 7 6) (TBS) containing 0-05% (v/v) Tween 20 (washing buffer), and incubated with 300 p1 of 0-15 M Tris-HCI (pH 7-6) containing I /. (w/v) bovine serum albumin (BSA) (blocking buffer) overnight at 4 C to block the free binding sites. Test serum samples (100 p,), which had been appropriately diluted (usually 1/400) with the blocking buffer, were placed in the wells and incubated overnight at 4'C. The subsequent procedures were carried out as described previously using peroxidase-labelled rabbit anti-human IgG for determination of IgG bound to the wells. For colorimetry, we used a micro ELISA reader (SLT210) at 486 nm. The antibody levels were expressed as optical density units (A 486) and tentatively termed the ELISA value. Anti-dsDNA antibody assay Detection of antibodies against double-stranded (ds) DNA or single-stranded (ss) DNA was performed by ELISA as described previously [8]. Polystyrene microtitre plates were precoated with poly-L-lysine (PLL) at room temperature for 30 min, followed by coating with dsDNA or ssDNA, and any residual binding sites were covered by 1% BSA-TB for I h. Diluted serum (1/400) was added to the microtitre plates followed by overnight incubation at 4--C. After washing, horseradish-peroxidase-labelled rabbit anti-human IgG was added, incubated for I h, and the enzyme substrate (ophenylenediamine) was then added to produce a chromogenic reaction. The following procedure was performed in a manner similar to that described in the case of anti-sulphatide antibodies.
439
Anti-heparan sulphate antibody assay Detection of antibodies against heparan sulphate (HS) was performed by ELISA as described previously [4]. Polystyrene microtitre plates were pre-coated with protamine chloride (0-5 mg/ml) at room temperature for 30 min, followed by coating with HS (100 pl, 2-5 pg/well; Seikagaku Kogyo, Tokyo, Japan). Thereafter the ELISA procedure was performed in a manner similar to that described in the case of anti-dsDNA antibodies. ELISA for dextran sulphate-binding IgG Detection of DXS-binding IgG was performed by ELISA in a manner similar to that used in the anti-dsDNA antibody assay. DXS was purchased from Sigma. Microtitre plates, pre-coated with 100 pi of PLL (0-4 pg/ml in TB), were coated for 30 min at room temperature with DXS (100 p1, 250 pg/ml in TB). Thereafter the ELISA procedure was performed in a manner similar to that described in the case of anti-dsDNA antibodies.
Anti-cardiolipin IgG antibody assay Detection of antibodies against cardiolipin was performed by ELISA in a manner similar to that used in the anti-dsDNA antibody assay. Cardiolipin (Sigma) in ethanol (50 pg/ml) was adsorbed onto the surface of polystyrene microtitre plates after evaporation of ethanol by overnight incubation in the dark, at 4"iC. Thereafter the ELISA procedure was performed in a manner similar to that described in the case of anti-dsDNA antibodies. Adsorption of anti-sulphatide antibodies by various ligands In the preliminary experiments, we tested the binding capacity of the various gels by changing the serum/adsorbent ratio. As a result, saturated adsorption was observed when we mixed 0-1 ml of 1/10 diluted serum samples containing high levels of antisulphatide antibody and more than 0-1 ml (packed volume) of the gels. Accordingly, in the following ligand adsorption experiments 0-1 ml of a 1/10 diluted serum sample from a patient with SLE and a patient with AI-CAH were mixed with 0-1 ml (packed volume) of cellulose gels affixed with various ligands for 2 h at room temperature with intermittent shaking. After centrifugation, supernatants were obtained and assayed for levels of antibodies against sulphatide. The levels of antisulphatide antibody remaining in the gel supernatants were estimated by plotting the optical density of the standards at 486 nm versus arbitrarily defined antibody units for the standards. It was possible to linearize the data using log/log paper and regression analysis was applied to the log transformation. The results were expressed as a percentage of adsorption of antibody levels in the supernatant of the control gel without ligands. As far as the reproducibility of the ligand adsorption experiment is concerned, the levels of anti-sulphatide antibody remaining in the gel supernatants after mixing the SLE serum and dextran sulphate gel were 7-8 +0-6 U/ml (n= 8).
Adsorption of anti-sulphatide antibodies by dextran sulphate gel chromatography A 01-ml serum sample from a patient with SLE was passed through dextran sulphate gel packed into a column (Sepacol Mini PP, Seikagaku Kogyo) and I-ml fractions were obtained. Subsequently, the column was washed thoroughly with TB and the bound materials were eluted with 1 5 M NaCl in TB, followed
S. Aotsuka et al.
440
by collection of -ml fractions. After dialysis overnight against TB, each fraction was tested for antibody activity against sulphatide, cardiolipin, dextran sulphate, dsDNA and ssDNA. DNAse treatment of serum samples Representative serum samples from patients with AI-CAH and SLE were each digested with DNAse I (Sigma). Briefly, 01 ml of each serum sample diluted 1/10 with PBS (pH 74, 015 M) -0 005 M MgCl2 (PBS-Mg) was mixed with 01 ml of DNAse I solution (1 mg/ml in PBS-Mg) and incubated for 60 min at 37°C, and digestion was then terminated by adding 0-2 ml of PBS (pH 7 4, 0-15 M)-0-01 M EDTA (PBS-EDTA). To provide a nondigested control, a serum sample was first mixed with 0-2 ml of PBS-EDTA and incubated, and then 01 ml of DNAse I solution was added to yield the same final composition.
mean serum level of anti-sulphatide antibodies in active SLE was significantly higher than in inactive SLE (t = 2-50, P < 005, paired t-test), other rheumatic diseases (t = 2-24, P < 0-05), and in healthy controls (t=3-00, P
Antibodies against sulphatide in sera from patients with autoimmune rheumatic diseases S. AOTSUKA, M. OKAWA-TAKATSUJI, S. UWATOKO, R. YOKOHARI*, Y. IKEDAt & G. TODAt Division of Immunology, Clinical Research Institute, National Medical Center, Tokyo, *National Atami Hospital, Shizuoka, and tFirst Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan
(Acceptedfor publication 3 October 1991)
SUMMARY We tested sera of patients with various autoimmune rheumatic diseases for the presence of antibodies against sulphatide (an acidic glycosphingolipid), identified as a target antigen for antibodies against the liver cell membrane. Thirty-five percent (7/20) of patients with lupus in the active stage possessed anti-sulphatide antibodies, whereas 10% (2/20) of those in the inactive stage and 20% (4/20) of those in the stationary stage possessed such antibodies. Moreover, 10% (3/29) of patients with other autoimmune rheumatic diseases also possessed anti-sulphatide antibodies. The level of antisulphatide antibodies was significantly correlated with the levels of anti-double-stranded (ds) DNA antibodies (r = 0-634, P < 0-001) and dextran sulphate-binding IgG (r = 0 407, P < 0 001). The serum levels of antibodies against sulphatide were correlated with a history of seizures or psychosis in patients with autoimmune rheumatic diseases. Gels coupled with polyanionic dextran sulphate, monoanionic sulphanilic acid and DNA were shown effectively to adsorb anti-sulphatide antibodies in the sera of patients with active systemic lupus erythematosus (SLE) and autoimmune chronic active hepatitis (Al-CAH). These results suggest that the observed reactivity with sulphatide is due to the presence of antibodies capable of reacting with various anionic molecules in the sera of patients with autoimmune rheumatic diseases as well as those with AI-CAH. Keywords sulphatide anti-dsDNA antibodies heparan sulphate dextran sulphate
INTRODUCTION Anti-DNA antibodies, which serve as significant diagnostic and prognostic markers in systemic lupus erythematosus (SLE), have been shown to have the capacity to react with a wide variety of molecules with repeating negative charges such as cardiolipin and other negatively charged phospholipids [1,2], chondriotin sulphate, heparan sulphate and Cibacron blue F3GA [3,4]. Autoimmune chronic active hepatitis (AI-CAH) is characterized by the presence of autoantibodies that react with various substances including nuclear constituents, smooth muscle cells, and hepatocyte plasma membranes. Recently, it was reported [5] that sera from patients with AT-CAH contain an IgG-class antibody against the acidic glycosphingolipid fraction of the rabbit hepatocyte plasma membranes. Antibodies to sulphatide were frequently found in sera from patients with AT-CAH but rarely in patients with virus-induced chronic active liver disease. The fact that sulphatide contains anionic sulphated residues prompted us to evaluate the antibody specificity and clinical
correlations of anti-sulphatide antibodies in autoimmune rheumatic diseases. PATIENTS AND METHODS Sera Sera from patients with active or inactive SLE (40 paired sera), stationary SLE (20 sera), other connective tissue diseases (29 serum samples: systemic sclerosis (n = 16); rheumatoid arthritis (n=4); mixed connective tissue disease (n=4); polymyositis (n = 3); Sjogren's syndrome (n = 2)), and autoimmune chronic active hepatitis (Al-CAH, 16 sera) were collected, stored at -80 C and thawed at 37°C before use. Al-CAH was diagnosed as described previously [6]. The clinical assessment of SLE disease activity was based on objective clinical disease manifestations and laboratory evidence of disease activity: (i) fever; (ii) arthralgia; (iii) cutaneous erythema; (iv) oral ulcer or alopecia; (v) elevated erythrocyte sedimentation rate (> 30 mm/h); (vi) hypocomplementaemia (CH50 < 20 U/ml or C3 < 60 mg/dl); (vii) leukopenia ( < 4 0 x 109/l); (viii) hypoalbuminaemia ( < 35 g/l); and (ix) positive LE cell or LE test. The activity of SLE was judged at the times of serum sampling. An arbitrary clinical assessment system for SLE disease activity yielded values
Correspondence: Shinichi Aotsuka, Clinical Research Institute, National Medical Center, 1-21-1, Toyama, Shinjuku-ku, Tokyo 162, Japan.
438
Anti-sulphatide antibody in autoimmune rheumatic diseases ranging from over four for active SLE to less than three for inactive or stationary SLE. Stationary SLE was defined as the absence of active disease, at least during our observation period. Based on previous experience, serum enzyme elevations were defined arbitrarily as follows: SGOT > 55 U/i (normal laboratory range 0-45) provided that myositis and haemolysis were absent, and/or SGPT >45 U/i (0-35), and/or ALP > 150 U/I (30-135) provided that the hepatic isoenzyme was demonstrated to be elevated. Patients were evaluated as to whether they had symptoms such as seizures or psychosis in the absence of offending drugs or known metabolic derangements.
Ligands Sorbent gels were prepared by coupling various molecules with different charges to cellulose gel for use as ligands. Dextran sulphate (DXS), purchased from Sigma Chemical Co., St Louis, MO, was immobilized via a hydroxyl group, and other ligands via their amino groups or the thiol group, according to the modified methods described by Vretblad [7]. Another anionic polymer (chondroitin sulphate, alginate), anionic monomers (pyromellitic acid, 2-aminoethyl hydrogen sulphate, sulphanilic acid, taurine), a cationic monomer (lysine), and a hydrophobic monomer (benzyl amine) were also coupled separately to the cellulose gel. DNA cellulose was purchased from Sigma. Anti-sulphatide antibody assay Fifty microlitres of sulphatide in ethanol (10 pg/ml) were placed in the wells of a microtitre plate (Nunc-Immunoplate I-96F; Nunc, Roskilde, Denmark) and evaporated overnight at 37 C. The wells were then washed six times with 0 135 M NaCl/0-0 1 5 M Tris-HCl (pH 7 6) (TBS) containing 0-05% (v/v) Tween 20 (washing buffer), and incubated with 300 p1 of 0-15 M Tris-HCI (pH 7-6) containing I /. (w/v) bovine serum albumin (BSA) (blocking buffer) overnight at 4 C to block the free binding sites. Test serum samples (100 p,), which had been appropriately diluted (usually 1/400) with the blocking buffer, were placed in the wells and incubated overnight at 4'C. The subsequent procedures were carried out as described previously using peroxidase-labelled rabbit anti-human IgG for determination of IgG bound to the wells. For colorimetry, we used a micro ELISA reader (SLT210) at 486 nm. The antibody levels were expressed as optical density units (A 486) and tentatively termed the ELISA value. Anti-dsDNA antibody assay Detection of antibodies against double-stranded (ds) DNA or single-stranded (ss) DNA was performed by ELISA as described previously [8]. Polystyrene microtitre plates were precoated with poly-L-lysine (PLL) at room temperature for 30 min, followed by coating with dsDNA or ssDNA, and any residual binding sites were covered by 1% BSA-TB for I h. Diluted serum (1/400) was added to the microtitre plates followed by overnight incubation at 4--C. After washing, horseradish-peroxidase-labelled rabbit anti-human IgG was added, incubated for I h, and the enzyme substrate (ophenylenediamine) was then added to produce a chromogenic reaction. The following procedure was performed in a manner similar to that described in the case of anti-sulphatide antibodies.
439
Anti-heparan sulphate antibody assay Detection of antibodies against heparan sulphate (HS) was performed by ELISA as described previously [4]. Polystyrene microtitre plates were pre-coated with protamine chloride (0-5 mg/ml) at room temperature for 30 min, followed by coating with HS (100 pl, 2-5 pg/well; Seikagaku Kogyo, Tokyo, Japan). Thereafter the ELISA procedure was performed in a manner similar to that described in the case of anti-dsDNA antibodies. ELISA for dextran sulphate-binding IgG Detection of DXS-binding IgG was performed by ELISA in a manner similar to that used in the anti-dsDNA antibody assay. DXS was purchased from Sigma. Microtitre plates, pre-coated with 100 pi of PLL (0-4 pg/ml in TB), were coated for 30 min at room temperature with DXS (100 p1, 250 pg/ml in TB). Thereafter the ELISA procedure was performed in a manner similar to that described in the case of anti-dsDNA antibodies.
Anti-cardiolipin IgG antibody assay Detection of antibodies against cardiolipin was performed by ELISA in a manner similar to that used in the anti-dsDNA antibody assay. Cardiolipin (Sigma) in ethanol (50 pg/ml) was adsorbed onto the surface of polystyrene microtitre plates after evaporation of ethanol by overnight incubation in the dark, at 4"iC. Thereafter the ELISA procedure was performed in a manner similar to that described in the case of anti-dsDNA antibodies. Adsorption of anti-sulphatide antibodies by various ligands In the preliminary experiments, we tested the binding capacity of the various gels by changing the serum/adsorbent ratio. As a result, saturated adsorption was observed when we mixed 0-1 ml of 1/10 diluted serum samples containing high levels of antisulphatide antibody and more than 0-1 ml (packed volume) of the gels. Accordingly, in the following ligand adsorption experiments 0-1 ml of a 1/10 diluted serum sample from a patient with SLE and a patient with AI-CAH were mixed with 0-1 ml (packed volume) of cellulose gels affixed with various ligands for 2 h at room temperature with intermittent shaking. After centrifugation, supernatants were obtained and assayed for levels of antibodies against sulphatide. The levels of antisulphatide antibody remaining in the gel supernatants were estimated by plotting the optical density of the standards at 486 nm versus arbitrarily defined antibody units for the standards. It was possible to linearize the data using log/log paper and regression analysis was applied to the log transformation. The results were expressed as a percentage of adsorption of antibody levels in the supernatant of the control gel without ligands. As far as the reproducibility of the ligand adsorption experiment is concerned, the levels of anti-sulphatide antibody remaining in the gel supernatants after mixing the SLE serum and dextran sulphate gel were 7-8 +0-6 U/ml (n= 8).
Adsorption of anti-sulphatide antibodies by dextran sulphate gel chromatography A 01-ml serum sample from a patient with SLE was passed through dextran sulphate gel packed into a column (Sepacol Mini PP, Seikagaku Kogyo) and I-ml fractions were obtained. Subsequently, the column was washed thoroughly with TB and the bound materials were eluted with 1 5 M NaCl in TB, followed
S. Aotsuka et al.
440
by collection of -ml fractions. After dialysis overnight against TB, each fraction was tested for antibody activity against sulphatide, cardiolipin, dextran sulphate, dsDNA and ssDNA. DNAse treatment of serum samples Representative serum samples from patients with AI-CAH and SLE were each digested with DNAse I (Sigma). Briefly, 01 ml of each serum sample diluted 1/10 with PBS (pH 74, 015 M) -0 005 M MgCl2 (PBS-Mg) was mixed with 01 ml of DNAse I solution (1 mg/ml in PBS-Mg) and incubated for 60 min at 37°C, and digestion was then terminated by adding 0-2 ml of PBS (pH 7 4, 0-15 M)-0-01 M EDTA (PBS-EDTA). To provide a nondigested control, a serum sample was first mixed with 0-2 ml of PBS-EDTA and incubated, and then 01 ml of DNAse I solution was added to yield the same final composition.
mean serum level of anti-sulphatide antibodies in active SLE was significantly higher than in inactive SLE (t = 2-50, P < 005, paired t-test), other rheumatic diseases (t = 2-24, P < 0-05), and in healthy controls (t=3-00, P
