Journal of Autoimmunity (1991) 4,819-830
Binding Profiles of Anticardiolipin Antibodies in Sera from Patients with SLE and Infectious Diseases
0. Vaarala Laboratory
of Clinical Immunology, National Helsinki, Finland
Public Health Institute,
(Received 25 January 1991 and accepted 16 April 1991) Inhibition experiments were performed to study the specificity of IgGclass antibody, binding to cardiolipin immobilized onto a polystyrene surface, in sera from patients with systemic lupus erythematosus (SLE) or infection. Six different phospholipids (three anionic: cardiolipin, phosphatidylserine and phosphatidic acid, and three neutral: phosphatidylcholine, phosphatidylethanolamine and platelet activating factor), lipopolysaccharide from Salmonella Minnesota (ReLPS), strain Re595 and lipoteichoic acid from Streptococcus pyogenes were used as inhibitors, in the form of liposomes. Eight of fifteen SLE sera exhibited strong reactivity to phosphatidylserine liposomes; other anionic phospholipids, cardiolipin and phosphatidic acid, were less effective inhibitors. The binding was inhibited effectively only by cardiolipin in three of the SLE sera, and by none of the anionic phospholipids tested in the remaining four SLE sera. In most sera from patients with bacterial infections (including syphilis), anticardiolipin antibodies (ACA) were inhibited only by cardiolipin, but in some cases also by phosphatidic acid. In Gram-negative infections, ACA were inhibited by ReLPS more effectively than by cardiolipin. ReLPS also inhibited ACA in two of five chlamydial sera. Appreciable inhibition of ACA by phosphatidylserine did not occur in infections. Thus, in contrast to previous studies, broad reactivity to anionic phospholipids occurred in only about half of SLE sera. This pattern of polyreactivity was not seen in infections.
Introduction Anticardiolipin antibodies (ACA) are a group of antiphospholipid antibodies which are frequently detected in the sera of patients with systemic lupus erythematosus Correspondence to: 0. Vaarala, Marmerheimintie 166, SF-00300 Helsinki, Finland. 819 0896-8411/91/050819+
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(SLE) or related autoimmune disorders, in many infections, and also in apparently healthy individuals [ 11. In traditional standard tests for syphilis, e.g. the Venereal Disease Research Laboratory (VDRL) slide flocculation test, the antigen is a mixture of cardiolipin, phosphatidylcholine and cholesterol. The anionic phospholipid, cardiolipin, is suggested as the antigenic component, whereas the neutral phospholipid, phosphatidylcholine and cholesterol are thought to be needed only for appropriate expression of the antigenic determinant(s) in cardiolipin [2]. Using solid phase immunoassays, introduced in 1983 [3], ACA have been found in 15 to 40% of patients with SLE; these antibodies have been reported to be associated with recurrent thrombosis, thrombocytopenia and spontaneous abortions, which are the clinical features of so-called ‘antiphospholipid syndrome’ [ 11. Only a small proportion of syphilitic sera with a positive VDRL test are positive in solid-phase immunoassays for ACA [4]. On the other hand, the majority of SLE sera with ACA are negative in the VDRL test [5,6]. It has been suggested that this is because the epitopes in cardiolipin for antibodies in SLE and syphilis are different [7]. Binding of immunoglobulins from SLE sera to solid-phase cardiolipin can be inhibited by liposomes containing anionic phospholipids, e.g. cardiolipin, phosphatidylserine or phosphatidic acid, but ACA found in syphilis are effectively inhibited only by cardiolipin and phosphatidic acid [4,8]. Furthermore, ACA from syphilitic sera are better inhibited by the antigen mixture used in the VDRL slide flocculation test than by pure cardiolipin liposomes which, in turn, are better inhibitors of the ACA in SLE sera [6,9]. ACA are also frequently found in many non-syphilitic infections [lO-161. We have reported that ACA in sera from patients with Gram-negative infections are effectively inhibited by lipopolysaccharide (LPS) from a rough strain of Salmonella minnesota [ 141. This pattern of apparent cross-reactivity was not seen in sera from patients with Gram-positive infections, syphilis or SLE. LPS is a common constituent of the outer membrane of Gram-negative bacteria and has structural similarity with cardiolipin. Accordingly, we have suggested that LPS may be the initiator antigen for cardiolipin-binding antibodies seen in Gram-negative infections. Only a minority of patients with ACA are subject to repeated episodes of venous and arterial thrombosis, repeated fetal loss or thrombocytopenia (‘antiphospholipid syndrome’) [ 11. This may be the result of differences in the antibody level, isotype distribution, avidity or specificity of ACA indifferent disorders. Infections represent one mechanism which has been shown to provoke the production of ACA. In SLE the origin of ACA is totally unknown. The specificity of ACA in SLE has been intensively studied recently, but little is known about the reactivity of infectionassociated ACA with different phospholipids. In this study IgG-class ACA in sera from patients with SLE and different infections were investigated by inhibition experiments in order to detect possible differences in the fine specificity of cardiolipin-binding antibodies detected by a solid-phase enzyme immunoassay. Materials and methods Patients The material for inhibition studies originated from two different groups of patients: 15 patients with SLE, as an example of an autoimmune disease, and 31 patients with
ACA in SLE and infections Table
821
1. Study materialfor inhibition experiments*
Disease SLET Infection: Syphilis1 Gram-positive infections Staphylococcal septicaemia Streptococcal septicaemia Gram-negative infections Salmonella Escherichiu coli septicaemia Chlamydial infection Infectious mononucleosis
Number of patients (n=46)
Inhibitors
15
CL, PA, PC, PE, I’S, ReLPS, PAF
6
CL, PA, PC, PE, PS, ReLPS CL, PA, PC, PE, PS, ReLPS, TA
5 5 3 2 5 5
CL, PA, PC, PE, PS, ReLPS CL, PA, PC, PE, PS, ReLPS CL, PA, PC, PE, PS, ReLPS
*CL = cardiolipin; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PS = phosphatidylserine; ReLPS = lipopolysaccharide; PAF = platelet activating factor; TA = teichoic acid. tPatients fulfilled the American Rheumatism Association’s revised classification criteria for SLE. fsyphilis was diagnosed by positive VDRL and Treponema pallidurn immobilization tests.
an infection (see Table 1). Patients having an elevated level of IgG-class ACA were selected for the study. Twelve of the 15 SLE patients had lupus anticoagulant (LA) detected by one of two different assays, recalcification time and the dilute Russel viper venom time as described previously [ 171. Antigens Cardiolipin in ethanol, phosphatidylethanolamine, phosphatidycholine, phosphatidylserine in chloroform, phosphatidic acid, platelet activating factor, lipopolysaccharide from Salmonella minnesota Re 595, and lipoteichoic acid from Streptococcuspyogenes were all obtained from Sigma Chemical Co. (St. Louis, MO, USA). Methods Antibodies to cardiolipin were detected by solid-phase enzyme-linked immunosorbent assay (ELISA) as previously described [14]. Briefly, polystyrene microtitre plates (Nunc, Roskilde, Denmark) were coated with cardiolipin at a concentration of 48 pg/ml in ethanol and left to dry overnight at 4°C. One percent human serum albumin (HSA) in phosphate-buffered saline (PBS) was used as blocking agent. The sera were studied in 0.2% HSA-PBS at a dilution of 1:50 for IgG and IgM class antibodies and 1:30 for IgA class antibodies. Alkaline-phosphatase-conjugated rabbit anti-human IgG, IgM or IgA (Jackson ImmunoResearch, West Grove, PA, USA) diluted in 0.2% HSA-PBS was used as the second antibody. The colour that developed with lmg/ml p-nitrophenyl phosphate (Sigma) in carbonate buffer, pH
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9.8, was read at 405 mm, using a Titertek Multiskan spectrophotometer (Eflab, Helsinki, Finland). The method has been standardized as defined by the International Workshop on Evaluation of the Anticardiolipin Test [18]. It was recently suggested that a plasma cofactor (&-glycoprotein I) is necessary for ACA to bind cardiolipin [ 19,201. This cofactor is claimed to be present in fetal calf serum, which is widely used as a blocking factor and supplement for dilution buffers in solid-phase immunoassays for ACA. In our cardiolipin ELISA we used HSA instead of fetal calf serum. HSA was separated from normal plasma by Cohn fractionation (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). To determine whether a similar cofactor is present in our HSA-PBS solution, serial dilutions of 11 sera (7 SLE and 4 syphilis sera) were studied using, in parallel, 10% fetal calf serum in PBS, HSA-PBS and 0.3% gelatin-PBS. The results were similar with the first two buffers, indicating that the cofactor or molecule(s) with similar functional properties is also present in our HSA preparation (data not shown). In contrast, the results were different when gelatin-PBS was used as the serum diluent. For the inhibition studies, antigens (see Table 1) were diluted in PBS at a concentration of 1 mg/ml and sonicated for 5 to 10min. The sera diluted 150 in 0.2% HSA-PBS with different concentrations of inhibitors (0.05 ug/ml to 50 ug/ml) were incubated overnight at room temperature, centrifuged, and the supernatants studied for IgG class ACA. The binding of antibodies to a neutral phospholipid was studied using phosphatidylcholine as a solid-phase antigen, at a concentration of 50 ug/ml in ethanol. The method was otherwise the same as for the anticardiolipin antibody assay. Results
SLE patients exhibited three different inhibition patterns of ACA. In eight of the 15 patients, the most effective inhibitor was phosphatidylserine when used at a low concentration (Figure la, b). Cardiolipin and phosphatidic acid inhibited the binding to a lesser extent. The three anionic phospholipids inhibited the binding in a similar manner (data not shown) but at a concentration 10 times higher. In three sera, the ACA activity was effectively inhibited only by cardiolipin (Figure lc). In the remaining four sera, none of the anionic phospholipids inhibited the cardiolipin binding. However, in one of them binding was inhibited to some extent by plateletactivating factor (Figure Id). The differences in the binding profile of ACA in SLE sera were not related to the antibody level (data not shown). ACA in all six syphilitic sera were effectively inhibited by cardiolipin (Figure 2a), and in two cases by phosphatidic acid (Figure 2b). Three of the patients with SLE had had multiple thrombosis, one of whom also had pulmonary emboli. There were no data available regarding the other clinical features of antiphospholipid syndrome. All three patients were LA-positive and had ACA with polyreactivity to anionic phospholipids tested (data not shown). This kind of polyreactivity was not seen in LA-negative SLE patients. Teichoic acid preparation was an ineffective inhibitor. In sera from patients with Gram-positive infections, the ACA were inhibited by cardiolipin and phosphatidic acid in three of five cases (Figure 2~). In the two remaining cases none of the antigens used inhibited binding to solid-phase cardiolipin.
CL
PS
PA
PC PE
ReLPS
Figure 1. Percentage see Table 1,
inhibition
Concentration
PA
PE
ReLPS
PAF
ACA in sera from four patients
50 pg/ml
of IgG-class
of inhibitor
PC
0
PS
0
CL
20
20
80
100
40
3
40
Patient
5 pg/ml
60
of inhibitor
0
60
Concentration
PAF
40
40
20
60
100
60
I 80
Patient
80
100
PS
PA
PA
with SLE by different
Concentration
PS
Concentration
L CL
CL
PC
PE
PE
inhibitors.
of inhibitor
PC
of inhibitor
PAF
PAF
4
2
For abbreviations
50 pg/ml
ReLPS
Patient
5pg/ml
ReLPS
Patient
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In all sera from patients with Gram-negative infections, LPS from Salmonella minnesota was a more effective inhibitor than cardiolipin or phosphatidic acid (Figure 2d). In one serum from a patient with salmonella infection, cardiolipin binding antibodies were inhibited by ReLPS but not by cardiolipin. Cardiolipin inhibited anticardiolipin binding in only one of the five sera from patients wth infectious mononucleosis (Figure 3a). No effect was observed with the other antigens. In sera from patients with chlamydial infection, the IgG-class ACA were inhibited by ReLPS in two of five cases (Figure 3b, c). In one of them binding to cardiolipin was also inhibited by cardiolipin-liposomes (Figure 3b). In three cases there was no inhibition by any of the antigens used. In the cases where inhibition was by any antigens tested not observed, IgG class binding to solid-phase phosphatidylcholine was analysed. No significant binding to phosphatidylcholine occurred in these sera (data not shown). Discussion
Previous studies have suggested that in autoimmune disorders, ACA are specific for anionic phospholipids in general [4,8,21,22]. It hasbeen shown that other anionic phospholipids, such as phosphatidylserine or phosphatidic acid, can replace cardiolipin in solid-phase assays [21]. In the present study phospholipid-liposomes were used to inhibit ACA detected by solid-phase enzyme immunoassay. The specificity of IgG-class ACA in patients with SLE proved to be heterogenous. In only half of the cases were ACA inhibited by all the negatively-charged phospholipids studied, a feature which indicates polyreactivity to anionic phospholipids. In SLE patients with broadly reactive ACA, phosphatidylserine liposomes proved to be the most effective inhibitor. Another antiphospholipid antibody, LA, often occurs together with ACA in sera from patients with SLE [ 11. These two antibodies have been suggested to have partially overlapping-specificities [4]. It has been suggested that LA is specific for phosphatidylserine [23], and a correlation between LA activity and binding to phosphatidylserine has been shown [24]. It has been proposed that antiphospholipid antibodies responsible for a positive LA test form a subset within the antiphospholipid antibody population which can be detected by anticardiolipin antibody assay [ 11. The strong reactivity of ACA with phosphatidylserine liposomes in certain SLE sera may be a factor determining anticoagulant activity. None of the three LA-negative SLE sera of the present study showed polyreactivity. Noticeable reactivity of ACA with phosphatidylserine did not occur in any serum from infected patients (including syphilis). The results indicate that ACA in syphilis and certain non-syphilitic infections have a high degree of structural specificity for cardiolipin-liposomes. In a previous study ACA in sera from patients with SLE were inhibited by liposomes containing phosphatidylserine or cardiolipin, in contrast to ACA in sera from patients with syphilis which were inhibited effectively only by cardiolipin liposomes [8]. The results of the present study are consistent with this observation, although in our material only half of the SLE sera were inhibited by phosphatidylserine. In their comprehensive study on syphilitic sera, Costello and Green showed that although syphilitic sera contained antibodies cross-reacting with cardiolipin,
PS
PC
PE
PA
Concentration
PS
PE
of inhibitor
PC 5Opg/ml
ReLPS
TA
-
PS
PS Concentration
CL
Concentration
CL
PC
PC of inhibitor
PA
of inhibitor
PA
PE
ReLPS
ReLPS 5 ,ug/ml
PE
Patient
50 pg/ml
8
Figure 2. Percentage inhibition of IgG-class ACA in sera from patients with syphilis (patients 5 and 6), Staphylococcal septicaemia (patient 7) and Salmonella infection (patient 8). For abbreviations see Table 1.
CL
0
80
100
0
7
20
Patient
ReLPS
20
50pg/ml
40
of inhibitor
PA
80
40
Concentration
CL
5
60
80 t
Patient
60
I””
Figure 3. Inhibition of IgG-class binding mononucleosis (IM), (patient 10) chlamydial
9
100
0.1 I
qRelps
Concentration
l CL
IO
---_
of inhibitor
r
(pg/ml)
Patient
0
-a
IO
100
to cardiolipin immobilized onto solid-phase in sera from patients infection, and (patient 11) chlamydial infection.
Patient
with different
0.1
(patient
IO
,a----_
infections:
I
.’
100
II
9) infectious
--a
Patient
ACA in SLE and infections
827
phosphatidic acid and phosphatidylserine immobilized onto nitrocellulose paper, the reactivity was almost always greater with cardiolipin than with the other anionic phospholipids [25]. On the basis of the results of stoichiometric calculations, they also suggested that ACA in syphilis do not recognize the individual cardiolipin molecule as the antigenic site, but instead recognize some structural form of the phospholipid. Taken together, the results of the above studies [8,25] and the present study indicate that ACA may be directed against different conformational epitopes in cardiolipin, and that the solid-phase assays are unable to detect these differences in the phospholipid antigens immobilized on a solid support. Changes in the configuration of phospholipids have been reported to play a role in both their antigenic and their immunogenic properties [26,27]. We have previously reported that the ACA present in sera from patients with different, mainly viral, infections cannot always be inhibited by cardiolipin liposomes [ 111. Interestingly, in four of the 15 SLE sera in this study, the same kind of reactivity to immobilized cardiolipin only, but not to cardiolipin as liposomes, was found. Many sera from patients with bacterial infections also showed this kind of reactivity. The binding cannot be considered non-specific, since there was no reactivity towards immobilized phosphatidylcholine used as a control antigen. The structure of the epitope(s) responsible for this type of binding to immobilized cardiolipin only is unknown. We have suggested that in Gram-negative infections, cardiolipin-binding antibodies may initially arise as a response to LPS of the outer membrane of Gramnegative bacteria [14]. The lipid A part of LPS contains negatively charged phosphate groups and fatty acids resembling the structure of cardiolipin. It has been shown that LPS of chlamydial species shares antigenic determinants with LPS from rough strains ofSalmonella minnesota and Salmonella typhimurium [28]. The short sugar residue and the lipid A part of the LPS molecule have been suggested as the shared determinants [29]. In the present study ReLPS effectively inhibited the binding of antibodies to cardiolipin in two of five chlamydial sera. This finding provides further evidence for the possible microbial origin of cardiolipin-binding antibodies in certain infections. It has been reported that in rabbits, lipoteichoic acid from Gram-positive bacteria elicited antibodies reacting with cardiolipin in the standard test for syphilis [30]. However, the lipoteichoic acid preparation used in the present study did not inhibit cardiolipin-binding antibodies in sera from patients with Gram-positive infections. Despite the frequent occurrence of ACA, ‘antiphospholipid syndrome’ is virtually never seen in patients with syphilis or other infections. It may be that ACA with restricted specificity to cardiolipin liposomes, which seem to occur not only in certain infections but also, as shown in the present study, in a subgroup of patients with SLE, are primarly induced by microbial antigens, such as LPS, during infections. Presumably, ACA of this pattern would be able to bind to antigenic determinants on microbial membranes but perhaps not to undamaged autologous structures. The production of broadly reactive ACA in SLE patients may be a consequence of dysregulation of an antigen-driven immune response. Recent studies have shown that ACA in some SLE sera bind to a complex of &,-glycoprotein I and cardiolipin [ 19,201. It may be that polyreactive ACA in SLE are directed against an antigenic complex which can be formed by different anionic phospholipids combined with p,glycoprotein. There is preliminary evidence that unlike SLE, infection-associated
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ACA bind directly to the cardiolipin antigen in the absence of P,-glycoprotein [31]. Thus, the antigenic determinant for ACA in infections differs from that in SLE. This is consistent with the finding that ACA in infections are not reactive with all anionic phospholipids, but rather are specific to cardiolipin. However, it must be emphasized that the binding profile of ACA in some SLE sera also resembles the profile seen in infections. In conclusion, results of the present study indicate a marked heterogeneity of ACA in SLE. This finding may be related to the fact that all SLE patients with ACA do not have ‘antiphospholipid syndrome’. All three SLE patients with a history of multiple thrombosis exhibited polyreactive ACA in the study material. Although the number of patients is too small for statistical analysis, this raises the possibility that the polyreactivity might contribute to the thrombotic tendency associated with ACA in patients with SLE. In contrast to previous reports, narrow reactivity to cardiolipin, as demonstrated by inhibition experiments, was also found to occur in a substantial proportion of patients with SLE. The latter pattern of reactivity is frequently &een not only in syphilis but also in other infections.
Acknowledgements I wish to thank Dr Marjatta Leirisalo-Repo, The Second Department of Medicine, Helsinki University Central Hospital, for serum samples from patients with SLE, Dr Ville Valtonen, Department of Bacteriology and Immunology, University of Helsinki, for the sera from patients with bacterial infections, and Dr Taneli Jouhikainen from the Finnish Red Cross Blood Transfusion Service for performing the lupus anticoagulant tests. This study was supported by the Rheumatism Research Foundation and the Orion Corporation Research Foundation.
References 1. Harris, E. N. and G. R. V. Hughes. 1989. Antiphospholipid antibodies. In Arthritis and Allied Conditions. D. J. MC Carty, ed. Lea & Febiger, Philadelphia, PA. pp. 1068-1079 2. Inoue, K. and S. Nojima. 1969. Immunochemical studies of phospholipids IV: The reactivities of antisera against natural cardiolipin and synthetic cardiolipin analogues containing antigens. Chem. Phys. Lipids 3: 70-77 3. Harris, E. N., M. L. Boey, C. G. Mackworth-Young, A. E. Gharavi, B. M. Patel, S. Loizou, and G. R. V. Hughes. 1983. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet ii: 1211-1214 4. Harris, E. N., A. E. Gharavi, S. Loizou, G. Derue, J. K. Chan, B. M. Patel, C. G. Mackworth-Young, C. C. BUM, and G. R. V. Hughes. 1985a. Crossreactivity of antiphospholipid antibodies. J.Clin. Lab. Immunol. 16: l-6 5. Koike, T., M. Sueishi, H. Funaki, H. Tomioka, and S. Yoshida. 1984. Antiphospholipid antibodies and biological false positive serological test for syphilis in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 56: 193-199 6. Harris, E. N., A. E. Gharavi, A. Tincani, J. K. H. Chan, H. Englert, P. Mantelli, F. Allegro, G. Ballestrieri, and G. R. V. Hughes. 1985b. Affinity purified anti-cardiolipin and anti-DNA antibodies. J. Clin. Lab. Immunol. 17: 155-162 C. 1990. Antiphospholipid antibodies: more than just a disease 7. Mackworth-Young, marker? Immunol. Today 2: 60-65
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8. Harris, E. N., A. E. Gharavi, G. D. Wasley, and G. R. V. Hughes. 1988. Use of enzymelinked immunosorbent assay and of inhibition studies to distinguish between antibodies to cardiolipin from patients with syphilis or autoimmune disorders. J. Infect. Dis. 1: 23-31 1989. 9. Mouritsen, S., M. Hoier-Madsen, A. Wiik, 0. Orum, and N. Strandberg-Pedersen. The specificity of anti-cardiolipin antibodies from syphilis patients and from patients with systemic lupus erythematosus. Clin. Exp. Zmmunol. 76: 178-183 10. Norberg, R., R. Thorstensson, K. Krook, G. Sturfelt, and 0. Nived. 1985. Antibodies against phospholipids (cardiolipin) in SLE. In Protides of the Biological Fluids. H. Peeters, ed. Pergamon Press, Oxford. pp. 325-329 11. Vaarala, O., M. Kleemola, T. Palosuo, and K. Aho. 1986. Anticardiolipin response in acute infections. Clin. Zmmunol. Zmmunopath. 41: 8-15 12. El-Roiey, A., 0. Sela, D. A. Isenberg, C. B. Colaco, R. C. Kennedy, and Y. Schoenfeld. 1987. The sera of patients with Klebsiella infections contain a common anti-DNA idiotype (16/16) Id and anti-polynucleotide activity. Clin. Exp. Zmmunol. 67: 507-515 13. Canoso, R. T., L. I. Zon, and J. E. Groopman. 1987. Anticardiolipin antibodies associated with HTLV-III infection. Br. J. Haematol. 65: 495-498 14. Vaaraia, O., M. Vaara, and T. Palosuo. 1988. Effective inhibition of cardiolipin-binding antibodies in Gram-negative infections by bacterial lipopolysaccharide. Stand. J. Zmmunol. 28: 607-612 15. Santiago, M. B., W. Cossermelli, M. F. Tuma, M. N. Pinto, and R. M. Oliveira. 1989. Anticardiolipin antibodies in patients with infectious diseases. Clin. Rheumatol. 1: 23-28 16. Misra, R., P. J. W. Venables, C. Plater-Zyberk, P. F. Watkins, and R. N. Maini. 1989. Anti-cardiolipin antibodies in infectious mononucleosis react with the membrane of activated lymphocytes. C&n. Exp. Zmmunol. 75: 35-4-O 17. Jouhikainen, T., R. Kekomiiki, M. Leirisalo-Repo, T. Backlund, and G. Myllyli. 1990. Platelet autoantibodies detected by immunoblotting in systemic lupus erythematosus: association with the lupus anticoagulant, and with history of thrombosis and thrombocytopenia. Eur. J. Haematdl. 44: 234-239 18. Harris, E. N., A. E. Gharavi, S. S. Patel, and G. R. V. Hughes. 1987. Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin. Exp. Zmmunol. 68: 215-222 19. McNeil, H. P., R. J. Simpson, C. N. Chesterman, and S. A. Krilis. 1990. Antiphospholipid antibodies are directed against a complex antigen that includes a lipidbinding inhibitor of coagulation: B,-glycoprotein I (apolipoprotein H). Proc. Nutl. Acad. Sci. USA 87: 41204124 20. Galli, M., I’. Comfurius, C. Maassen, H. C. Hemker, M. H. de Baets, P. J. C. van BredaVriesman, T. Barbui, R. F. A. Zwaal, and E. M. Bevers. 1990. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet ii: 1544-1547 21. Gharavi, A. E., E. N. Harris, R. A. Asherson, and G. R. V. Hughes. 1987. Anticardiolipin antibodies: isotype distribution and phospholipid specificity. Ann. Rheum. Dis. 46: l-6 22. Loizou, S., C. G. Mackworth-Young, C. Cofiner, and M. J. Walport. 1990. Heterogeneity of binding reactivity to different phospholipids of antibodies from patients with systemic lupus erythematosus (SLE) and with syphilis. Clin. Exp. Zmmunol. 80: 171-176 23. Kelsey, P. R., K. J. Stevenson, and L. Poller. 1984. The diagnosis of lupus anticoagulants by the Activated Partial Thromboplastin Time - the central role of phosphatidyl serine. Thromb. Haemost. 52: 172-175 24. Branch, D. W., N. S. Rote, D. A. Dostal, and J. R. Scott. 1987. Association of lupus anticoagulant with antibody against phosphatidyl serine. Clin. Zmmunol. Zmmunopath. 42: 63-75 25. Costello, P. B. and F. A. Green. 1986. Reactivity patterns of human anticardiolipin and other antiphospholipid antibodies in syphilitic sera. Infect. Zmmun. 3: 771-775 26. Janoff, A. S. and J. Rauch. 1986: The structural specificity of antiphospholipid antibodies in autoimmune disease. Chsm. Phys. Lipids 40: 315-332 27. Rauch, J. and A. S. Janoff. 1990. Phospholipid in the hexagonal II phase is immunogenic: Evidence for immunorecognition of nonbilayer lipid phases in vivo. Proc. Natl. Acad. Sci. USA87:4112-4114
830 O.Vaarala 28. Nurminen, M., M. Leinonen, P. Saikku, and P. H. Miikeli. 1983. The genus-specific antigen of Chlamydia: resemblance to the polysaccharide of enteric bacteria. Science 220: 1279-1281 29. Caldwell, H. D. and P. J. Hitchcock. 1984. Monoclonal antibody against a genus-specific antigen of Chlamydia species: location of the epitope on chlamydial lipopolysaccharide. Infect. Immun. 2: 306-314 30. Wicken, A. J., J. W. Gibbens, and K. W. Knox. 1972. Anti-teichoic acid antibodies and non-treponemal serological tests for syphilis. Infect. Immun. 6: 982-984 31. McNeil, H. P., J. E. Hunt, and S. A. Krilis. 1990. New aspects of anticardiolipin antibodies. Clin. Exp. Rheumatol. 8: 525-527
Binding Profiles of Anticardiolipin Antibodies in Sera from Patients with SLE and Infectious Diseases
0. Vaarala Laboratory
of Clinical Immunology, National Helsinki, Finland
Public Health Institute,
(Received 25 January 1991 and accepted 16 April 1991) Inhibition experiments were performed to study the specificity of IgGclass antibody, binding to cardiolipin immobilized onto a polystyrene surface, in sera from patients with systemic lupus erythematosus (SLE) or infection. Six different phospholipids (three anionic: cardiolipin, phosphatidylserine and phosphatidic acid, and three neutral: phosphatidylcholine, phosphatidylethanolamine and platelet activating factor), lipopolysaccharide from Salmonella Minnesota (ReLPS), strain Re595 and lipoteichoic acid from Streptococcus pyogenes were used as inhibitors, in the form of liposomes. Eight of fifteen SLE sera exhibited strong reactivity to phosphatidylserine liposomes; other anionic phospholipids, cardiolipin and phosphatidic acid, were less effective inhibitors. The binding was inhibited effectively only by cardiolipin in three of the SLE sera, and by none of the anionic phospholipids tested in the remaining four SLE sera. In most sera from patients with bacterial infections (including syphilis), anticardiolipin antibodies (ACA) were inhibited only by cardiolipin, but in some cases also by phosphatidic acid. In Gram-negative infections, ACA were inhibited by ReLPS more effectively than by cardiolipin. ReLPS also inhibited ACA in two of five chlamydial sera. Appreciable inhibition of ACA by phosphatidylserine did not occur in infections. Thus, in contrast to previous studies, broad reactivity to anionic phospholipids occurred in only about half of SLE sera. This pattern of polyreactivity was not seen in infections.
Introduction Anticardiolipin antibodies (ACA) are a group of antiphospholipid antibodies which are frequently detected in the sera of patients with systemic lupus erythematosus Correspondence to: 0. Vaarala, Marmerheimintie 166, SF-00300 Helsinki, Finland. 819 0896-8411/91/050819+
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0 1991 Academic Press Limited
820
0. Vaarala
(SLE) or related autoimmune disorders, in many infections, and also in apparently healthy individuals [ 11. In traditional standard tests for syphilis, e.g. the Venereal Disease Research Laboratory (VDRL) slide flocculation test, the antigen is a mixture of cardiolipin, phosphatidylcholine and cholesterol. The anionic phospholipid, cardiolipin, is suggested as the antigenic component, whereas the neutral phospholipid, phosphatidylcholine and cholesterol are thought to be needed only for appropriate expression of the antigenic determinant(s) in cardiolipin [2]. Using solid phase immunoassays, introduced in 1983 [3], ACA have been found in 15 to 40% of patients with SLE; these antibodies have been reported to be associated with recurrent thrombosis, thrombocytopenia and spontaneous abortions, which are the clinical features of so-called ‘antiphospholipid syndrome’ [ 11. Only a small proportion of syphilitic sera with a positive VDRL test are positive in solid-phase immunoassays for ACA [4]. On the other hand, the majority of SLE sera with ACA are negative in the VDRL test [5,6]. It has been suggested that this is because the epitopes in cardiolipin for antibodies in SLE and syphilis are different [7]. Binding of immunoglobulins from SLE sera to solid-phase cardiolipin can be inhibited by liposomes containing anionic phospholipids, e.g. cardiolipin, phosphatidylserine or phosphatidic acid, but ACA found in syphilis are effectively inhibited only by cardiolipin and phosphatidic acid [4,8]. Furthermore, ACA from syphilitic sera are better inhibited by the antigen mixture used in the VDRL slide flocculation test than by pure cardiolipin liposomes which, in turn, are better inhibitors of the ACA in SLE sera [6,9]. ACA are also frequently found in many non-syphilitic infections [lO-161. We have reported that ACA in sera from patients with Gram-negative infections are effectively inhibited by lipopolysaccharide (LPS) from a rough strain of Salmonella minnesota [ 141. This pattern of apparent cross-reactivity was not seen in sera from patients with Gram-positive infections, syphilis or SLE. LPS is a common constituent of the outer membrane of Gram-negative bacteria and has structural similarity with cardiolipin. Accordingly, we have suggested that LPS may be the initiator antigen for cardiolipin-binding antibodies seen in Gram-negative infections. Only a minority of patients with ACA are subject to repeated episodes of venous and arterial thrombosis, repeated fetal loss or thrombocytopenia (‘antiphospholipid syndrome’) [ 11. This may be the result of differences in the antibody level, isotype distribution, avidity or specificity of ACA indifferent disorders. Infections represent one mechanism which has been shown to provoke the production of ACA. In SLE the origin of ACA is totally unknown. The specificity of ACA in SLE has been intensively studied recently, but little is known about the reactivity of infectionassociated ACA with different phospholipids. In this study IgG-class ACA in sera from patients with SLE and different infections were investigated by inhibition experiments in order to detect possible differences in the fine specificity of cardiolipin-binding antibodies detected by a solid-phase enzyme immunoassay. Materials and methods Patients The material for inhibition studies originated from two different groups of patients: 15 patients with SLE, as an example of an autoimmune disease, and 31 patients with
ACA in SLE and infections Table
821
1. Study materialfor inhibition experiments*
Disease SLET Infection: Syphilis1 Gram-positive infections Staphylococcal septicaemia Streptococcal septicaemia Gram-negative infections Salmonella Escherichiu coli septicaemia Chlamydial infection Infectious mononucleosis
Number of patients (n=46)
Inhibitors
15
CL, PA, PC, PE, I’S, ReLPS, PAF
6
CL, PA, PC, PE, PS, ReLPS CL, PA, PC, PE, PS, ReLPS, TA
5 5 3 2 5 5
CL, PA, PC, PE, PS, ReLPS CL, PA, PC, PE, PS, ReLPS CL, PA, PC, PE, PS, ReLPS
*CL = cardiolipin; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PS = phosphatidylserine; ReLPS = lipopolysaccharide; PAF = platelet activating factor; TA = teichoic acid. tPatients fulfilled the American Rheumatism Association’s revised classification criteria for SLE. fsyphilis was diagnosed by positive VDRL and Treponema pallidurn immobilization tests.
an infection (see Table 1). Patients having an elevated level of IgG-class ACA were selected for the study. Twelve of the 15 SLE patients had lupus anticoagulant (LA) detected by one of two different assays, recalcification time and the dilute Russel viper venom time as described previously [ 171. Antigens Cardiolipin in ethanol, phosphatidylethanolamine, phosphatidycholine, phosphatidylserine in chloroform, phosphatidic acid, platelet activating factor, lipopolysaccharide from Salmonella minnesota Re 595, and lipoteichoic acid from Streptococcuspyogenes were all obtained from Sigma Chemical Co. (St. Louis, MO, USA). Methods Antibodies to cardiolipin were detected by solid-phase enzyme-linked immunosorbent assay (ELISA) as previously described [14]. Briefly, polystyrene microtitre plates (Nunc, Roskilde, Denmark) were coated with cardiolipin at a concentration of 48 pg/ml in ethanol and left to dry overnight at 4°C. One percent human serum albumin (HSA) in phosphate-buffered saline (PBS) was used as blocking agent. The sera were studied in 0.2% HSA-PBS at a dilution of 1:50 for IgG and IgM class antibodies and 1:30 for IgA class antibodies. Alkaline-phosphatase-conjugated rabbit anti-human IgG, IgM or IgA (Jackson ImmunoResearch, West Grove, PA, USA) diluted in 0.2% HSA-PBS was used as the second antibody. The colour that developed with lmg/ml p-nitrophenyl phosphate (Sigma) in carbonate buffer, pH
822 0. Vaarala
9.8, was read at 405 mm, using a Titertek Multiskan spectrophotometer (Eflab, Helsinki, Finland). The method has been standardized as defined by the International Workshop on Evaluation of the Anticardiolipin Test [18]. It was recently suggested that a plasma cofactor (&-glycoprotein I) is necessary for ACA to bind cardiolipin [ 19,201. This cofactor is claimed to be present in fetal calf serum, which is widely used as a blocking factor and supplement for dilution buffers in solid-phase immunoassays for ACA. In our cardiolipin ELISA we used HSA instead of fetal calf serum. HSA was separated from normal plasma by Cohn fractionation (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). To determine whether a similar cofactor is present in our HSA-PBS solution, serial dilutions of 11 sera (7 SLE and 4 syphilis sera) were studied using, in parallel, 10% fetal calf serum in PBS, HSA-PBS and 0.3% gelatin-PBS. The results were similar with the first two buffers, indicating that the cofactor or molecule(s) with similar functional properties is also present in our HSA preparation (data not shown). In contrast, the results were different when gelatin-PBS was used as the serum diluent. For the inhibition studies, antigens (see Table 1) were diluted in PBS at a concentration of 1 mg/ml and sonicated for 5 to 10min. The sera diluted 150 in 0.2% HSA-PBS with different concentrations of inhibitors (0.05 ug/ml to 50 ug/ml) were incubated overnight at room temperature, centrifuged, and the supernatants studied for IgG class ACA. The binding of antibodies to a neutral phospholipid was studied using phosphatidylcholine as a solid-phase antigen, at a concentration of 50 ug/ml in ethanol. The method was otherwise the same as for the anticardiolipin antibody assay. Results
SLE patients exhibited three different inhibition patterns of ACA. In eight of the 15 patients, the most effective inhibitor was phosphatidylserine when used at a low concentration (Figure la, b). Cardiolipin and phosphatidic acid inhibited the binding to a lesser extent. The three anionic phospholipids inhibited the binding in a similar manner (data not shown) but at a concentration 10 times higher. In three sera, the ACA activity was effectively inhibited only by cardiolipin (Figure lc). In the remaining four sera, none of the anionic phospholipids inhibited the cardiolipin binding. However, in one of them binding was inhibited to some extent by plateletactivating factor (Figure Id). The differences in the binding profile of ACA in SLE sera were not related to the antibody level (data not shown). ACA in all six syphilitic sera were effectively inhibited by cardiolipin (Figure 2a), and in two cases by phosphatidic acid (Figure 2b). Three of the patients with SLE had had multiple thrombosis, one of whom also had pulmonary emboli. There were no data available regarding the other clinical features of antiphospholipid syndrome. All three patients were LA-positive and had ACA with polyreactivity to anionic phospholipids tested (data not shown). This kind of polyreactivity was not seen in LA-negative SLE patients. Teichoic acid preparation was an ineffective inhibitor. In sera from patients with Gram-positive infections, the ACA were inhibited by cardiolipin and phosphatidic acid in three of five cases (Figure 2~). In the two remaining cases none of the antigens used inhibited binding to solid-phase cardiolipin.
CL
PS
PA
PC PE
ReLPS
Figure 1. Percentage see Table 1,
inhibition
Concentration
PA
PE
ReLPS
PAF
ACA in sera from four patients
50 pg/ml
of IgG-class
of inhibitor
PC
0
PS
0
CL
20
20
80
100
40
3
40
Patient
5 pg/ml
60
of inhibitor
0
60
Concentration
PAF
40
40
20
60
100
60
I 80
Patient
80
100
PS
PA
PA
with SLE by different
Concentration
PS
Concentration
L CL
CL
PC
PE
PE
inhibitors.
of inhibitor
PC
of inhibitor
PAF
PAF
4
2
For abbreviations
50 pg/ml
ReLPS
Patient
5pg/ml
ReLPS
Patient
824 0. Vaarala
In all sera from patients with Gram-negative infections, LPS from Salmonella minnesota was a more effective inhibitor than cardiolipin or phosphatidic acid (Figure 2d). In one serum from a patient with salmonella infection, cardiolipin binding antibodies were inhibited by ReLPS but not by cardiolipin. Cardiolipin inhibited anticardiolipin binding in only one of the five sera from patients wth infectious mononucleosis (Figure 3a). No effect was observed with the other antigens. In sera from patients with chlamydial infection, the IgG-class ACA were inhibited by ReLPS in two of five cases (Figure 3b, c). In one of them binding to cardiolipin was also inhibited by cardiolipin-liposomes (Figure 3b). In three cases there was no inhibition by any of the antigens used. In the cases where inhibition was by any antigens tested not observed, IgG class binding to solid-phase phosphatidylcholine was analysed. No significant binding to phosphatidylcholine occurred in these sera (data not shown). Discussion
Previous studies have suggested that in autoimmune disorders, ACA are specific for anionic phospholipids in general [4,8,21,22]. It hasbeen shown that other anionic phospholipids, such as phosphatidylserine or phosphatidic acid, can replace cardiolipin in solid-phase assays [21]. In the present study phospholipid-liposomes were used to inhibit ACA detected by solid-phase enzyme immunoassay. The specificity of IgG-class ACA in patients with SLE proved to be heterogenous. In only half of the cases were ACA inhibited by all the negatively-charged phospholipids studied, a feature which indicates polyreactivity to anionic phospholipids. In SLE patients with broadly reactive ACA, phosphatidylserine liposomes proved to be the most effective inhibitor. Another antiphospholipid antibody, LA, often occurs together with ACA in sera from patients with SLE [ 11. These two antibodies have been suggested to have partially overlapping-specificities [4]. It has been suggested that LA is specific for phosphatidylserine [23], and a correlation between LA activity and binding to phosphatidylserine has been shown [24]. It has been proposed that antiphospholipid antibodies responsible for a positive LA test form a subset within the antiphospholipid antibody population which can be detected by anticardiolipin antibody assay [ 11. The strong reactivity of ACA with phosphatidylserine liposomes in certain SLE sera may be a factor determining anticoagulant activity. None of the three LA-negative SLE sera of the present study showed polyreactivity. Noticeable reactivity of ACA with phosphatidylserine did not occur in any serum from infected patients (including syphilis). The results indicate that ACA in syphilis and certain non-syphilitic infections have a high degree of structural specificity for cardiolipin-liposomes. In a previous study ACA in sera from patients with SLE were inhibited by liposomes containing phosphatidylserine or cardiolipin, in contrast to ACA in sera from patients with syphilis which were inhibited effectively only by cardiolipin liposomes [8]. The results of the present study are consistent with this observation, although in our material only half of the SLE sera were inhibited by phosphatidylserine. In their comprehensive study on syphilitic sera, Costello and Green showed that although syphilitic sera contained antibodies cross-reacting with cardiolipin,
PS
PC
PE
PA
Concentration
PS
PE
of inhibitor
PC 5Opg/ml
ReLPS
TA
-
PS
PS Concentration
CL
Concentration
CL
PC
PC of inhibitor
PA
of inhibitor
PA
PE
ReLPS
ReLPS 5 ,ug/ml
PE
Patient
50 pg/ml
8
Figure 2. Percentage inhibition of IgG-class ACA in sera from patients with syphilis (patients 5 and 6), Staphylococcal septicaemia (patient 7) and Salmonella infection (patient 8). For abbreviations see Table 1.
CL
0
80
100
0
7
20
Patient
ReLPS
20
50pg/ml
40
of inhibitor
PA
80
40
Concentration
CL
5
60
80 t
Patient
60
I””
Figure 3. Inhibition of IgG-class binding mononucleosis (IM), (patient 10) chlamydial
9
100
0.1 I
qRelps
Concentration
l CL
IO
---_
of inhibitor
r
(pg/ml)
Patient
0
-a
IO
100
to cardiolipin immobilized onto solid-phase in sera from patients infection, and (patient 11) chlamydial infection.
Patient
with different
0.1
(patient
IO
,a----_
infections:
I
.’
100
II
9) infectious
--a
Patient
ACA in SLE and infections
827
phosphatidic acid and phosphatidylserine immobilized onto nitrocellulose paper, the reactivity was almost always greater with cardiolipin than with the other anionic phospholipids [25]. On the basis of the results of stoichiometric calculations, they also suggested that ACA in syphilis do not recognize the individual cardiolipin molecule as the antigenic site, but instead recognize some structural form of the phospholipid. Taken together, the results of the above studies [8,25] and the present study indicate that ACA may be directed against different conformational epitopes in cardiolipin, and that the solid-phase assays are unable to detect these differences in the phospholipid antigens immobilized on a solid support. Changes in the configuration of phospholipids have been reported to play a role in both their antigenic and their immunogenic properties [26,27]. We have previously reported that the ACA present in sera from patients with different, mainly viral, infections cannot always be inhibited by cardiolipin liposomes [ 111. Interestingly, in four of the 15 SLE sera in this study, the same kind of reactivity to immobilized cardiolipin only, but not to cardiolipin as liposomes, was found. Many sera from patients with bacterial infections also showed this kind of reactivity. The binding cannot be considered non-specific, since there was no reactivity towards immobilized phosphatidylcholine used as a control antigen. The structure of the epitope(s) responsible for this type of binding to immobilized cardiolipin only is unknown. We have suggested that in Gram-negative infections, cardiolipin-binding antibodies may initially arise as a response to LPS of the outer membrane of Gramnegative bacteria [14]. The lipid A part of LPS contains negatively charged phosphate groups and fatty acids resembling the structure of cardiolipin. It has been shown that LPS of chlamydial species shares antigenic determinants with LPS from rough strains ofSalmonella minnesota and Salmonella typhimurium [28]. The short sugar residue and the lipid A part of the LPS molecule have been suggested as the shared determinants [29]. In the present study ReLPS effectively inhibited the binding of antibodies to cardiolipin in two of five chlamydial sera. This finding provides further evidence for the possible microbial origin of cardiolipin-binding antibodies in certain infections. It has been reported that in rabbits, lipoteichoic acid from Gram-positive bacteria elicited antibodies reacting with cardiolipin in the standard test for syphilis [30]. However, the lipoteichoic acid preparation used in the present study did not inhibit cardiolipin-binding antibodies in sera from patients with Gram-positive infections. Despite the frequent occurrence of ACA, ‘antiphospholipid syndrome’ is virtually never seen in patients with syphilis or other infections. It may be that ACA with restricted specificity to cardiolipin liposomes, which seem to occur not only in certain infections but also, as shown in the present study, in a subgroup of patients with SLE, are primarly induced by microbial antigens, such as LPS, during infections. Presumably, ACA of this pattern would be able to bind to antigenic determinants on microbial membranes but perhaps not to undamaged autologous structures. The production of broadly reactive ACA in SLE patients may be a consequence of dysregulation of an antigen-driven immune response. Recent studies have shown that ACA in some SLE sera bind to a complex of &,-glycoprotein I and cardiolipin [ 19,201. It may be that polyreactive ACA in SLE are directed against an antigenic complex which can be formed by different anionic phospholipids combined with p,glycoprotein. There is preliminary evidence that unlike SLE, infection-associated
828
0. Vaarala
ACA bind directly to the cardiolipin antigen in the absence of P,-glycoprotein [31]. Thus, the antigenic determinant for ACA in infections differs from that in SLE. This is consistent with the finding that ACA in infections are not reactive with all anionic phospholipids, but rather are specific to cardiolipin. However, it must be emphasized that the binding profile of ACA in some SLE sera also resembles the profile seen in infections. In conclusion, results of the present study indicate a marked heterogeneity of ACA in SLE. This finding may be related to the fact that all SLE patients with ACA do not have ‘antiphospholipid syndrome’. All three SLE patients with a history of multiple thrombosis exhibited polyreactive ACA in the study material. Although the number of patients is too small for statistical analysis, this raises the possibility that the polyreactivity might contribute to the thrombotic tendency associated with ACA in patients with SLE. In contrast to previous reports, narrow reactivity to cardiolipin, as demonstrated by inhibition experiments, was also found to occur in a substantial proportion of patients with SLE. The latter pattern of reactivity is frequently &een not only in syphilis but also in other infections.
Acknowledgements I wish to thank Dr Marjatta Leirisalo-Repo, The Second Department of Medicine, Helsinki University Central Hospital, for serum samples from patients with SLE, Dr Ville Valtonen, Department of Bacteriology and Immunology, University of Helsinki, for the sera from patients with bacterial infections, and Dr Taneli Jouhikainen from the Finnish Red Cross Blood Transfusion Service for performing the lupus anticoagulant tests. This study was supported by the Rheumatism Research Foundation and the Orion Corporation Research Foundation.
References 1. Harris, E. N. and G. R. V. Hughes. 1989. Antiphospholipid antibodies. In Arthritis and Allied Conditions. D. J. MC Carty, ed. Lea & Febiger, Philadelphia, PA. pp. 1068-1079 2. Inoue, K. and S. Nojima. 1969. Immunochemical studies of phospholipids IV: The reactivities of antisera against natural cardiolipin and synthetic cardiolipin analogues containing antigens. Chem. Phys. Lipids 3: 70-77 3. Harris, E. N., M. L. Boey, C. G. Mackworth-Young, A. E. Gharavi, B. M. Patel, S. Loizou, and G. R. V. Hughes. 1983. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet ii: 1211-1214 4. Harris, E. N., A. E. Gharavi, S. Loizou, G. Derue, J. K. Chan, B. M. Patel, C. G. Mackworth-Young, C. C. BUM, and G. R. V. Hughes. 1985a. Crossreactivity of antiphospholipid antibodies. J.Clin. Lab. Immunol. 16: l-6 5. Koike, T., M. Sueishi, H. Funaki, H. Tomioka, and S. Yoshida. 1984. Antiphospholipid antibodies and biological false positive serological test for syphilis in patients with systemic lupus erythematosus. Clin. Exp. Immunol. 56: 193-199 6. Harris, E. N., A. E. Gharavi, A. Tincani, J. K. H. Chan, H. Englert, P. Mantelli, F. Allegro, G. Ballestrieri, and G. R. V. Hughes. 1985b. Affinity purified anti-cardiolipin and anti-DNA antibodies. J. Clin. Lab. Immunol. 17: 155-162 C. 1990. Antiphospholipid antibodies: more than just a disease 7. Mackworth-Young, marker? Immunol. Today 2: 60-65
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8. Harris, E. N., A. E. Gharavi, G. D. Wasley, and G. R. V. Hughes. 1988. Use of enzymelinked immunosorbent assay and of inhibition studies to distinguish between antibodies to cardiolipin from patients with syphilis or autoimmune disorders. J. Infect. Dis. 1: 23-31 1989. 9. Mouritsen, S., M. Hoier-Madsen, A. Wiik, 0. Orum, and N. Strandberg-Pedersen. The specificity of anti-cardiolipin antibodies from syphilis patients and from patients with systemic lupus erythematosus. Clin. Exp. Zmmunol. 76: 178-183 10. Norberg, R., R. Thorstensson, K. Krook, G. Sturfelt, and 0. Nived. 1985. Antibodies against phospholipids (cardiolipin) in SLE. In Protides of the Biological Fluids. H. Peeters, ed. Pergamon Press, Oxford. pp. 325-329 11. Vaarala, O., M. Kleemola, T. Palosuo, and K. Aho. 1986. Anticardiolipin response in acute infections. Clin. Zmmunol. Zmmunopath. 41: 8-15 12. El-Roiey, A., 0. Sela, D. A. Isenberg, C. B. Colaco, R. C. Kennedy, and Y. Schoenfeld. 1987. The sera of patients with Klebsiella infections contain a common anti-DNA idiotype (16/16) Id and anti-polynucleotide activity. Clin. Exp. Zmmunol. 67: 507-515 13. Canoso, R. T., L. I. Zon, and J. E. Groopman. 1987. Anticardiolipin antibodies associated with HTLV-III infection. Br. J. Haematol. 65: 495-498 14. Vaaraia, O., M. Vaara, and T. Palosuo. 1988. Effective inhibition of cardiolipin-binding antibodies in Gram-negative infections by bacterial lipopolysaccharide. Stand. J. Zmmunol. 28: 607-612 15. Santiago, M. B., W. Cossermelli, M. F. Tuma, M. N. Pinto, and R. M. Oliveira. 1989. Anticardiolipin antibodies in patients with infectious diseases. Clin. Rheumatol. 1: 23-28 16. Misra, R., P. J. W. Venables, C. Plater-Zyberk, P. F. Watkins, and R. N. Maini. 1989. Anti-cardiolipin antibodies in infectious mononucleosis react with the membrane of activated lymphocytes. C&n. Exp. Zmmunol. 75: 35-4-O 17. Jouhikainen, T., R. Kekomiiki, M. Leirisalo-Repo, T. Backlund, and G. Myllyli. 1990. Platelet autoantibodies detected by immunoblotting in systemic lupus erythematosus: association with the lupus anticoagulant, and with history of thrombosis and thrombocytopenia. Eur. J. Haematdl. 44: 234-239 18. Harris, E. N., A. E. Gharavi, S. S. Patel, and G. R. V. Hughes. 1987. Evaluation of the anti-cardiolipin antibody test: report of an international workshop held 4 April 1986. Clin. Exp. Zmmunol. 68: 215-222 19. McNeil, H. P., R. J. Simpson, C. N. Chesterman, and S. A. Krilis. 1990. Antiphospholipid antibodies are directed against a complex antigen that includes a lipidbinding inhibitor of coagulation: B,-glycoprotein I (apolipoprotein H). Proc. Nutl. Acad. Sci. USA 87: 41204124 20. Galli, M., I’. Comfurius, C. Maassen, H. C. Hemker, M. H. de Baets, P. J. C. van BredaVriesman, T. Barbui, R. F. A. Zwaal, and E. M. Bevers. 1990. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet ii: 1544-1547 21. Gharavi, A. E., E. N. Harris, R. A. Asherson, and G. R. V. Hughes. 1987. Anticardiolipin antibodies: isotype distribution and phospholipid specificity. Ann. Rheum. Dis. 46: l-6 22. Loizou, S., C. G. Mackworth-Young, C. Cofiner, and M. J. Walport. 1990. Heterogeneity of binding reactivity to different phospholipids of antibodies from patients with systemic lupus erythematosus (SLE) and with syphilis. Clin. Exp. Zmmunol. 80: 171-176 23. Kelsey, P. R., K. J. Stevenson, and L. Poller. 1984. The diagnosis of lupus anticoagulants by the Activated Partial Thromboplastin Time - the central role of phosphatidyl serine. Thromb. Haemost. 52: 172-175 24. Branch, D. W., N. S. Rote, D. A. Dostal, and J. R. Scott. 1987. Association of lupus anticoagulant with antibody against phosphatidyl serine. Clin. Zmmunol. Zmmunopath. 42: 63-75 25. Costello, P. B. and F. A. Green. 1986. Reactivity patterns of human anticardiolipin and other antiphospholipid antibodies in syphilitic sera. Infect. Zmmun. 3: 771-775 26. Janoff, A. S. and J. Rauch. 1986: The structural specificity of antiphospholipid antibodies in autoimmune disease. Chsm. Phys. Lipids 40: 315-332 27. Rauch, J. and A. S. Janoff. 1990. Phospholipid in the hexagonal II phase is immunogenic: Evidence for immunorecognition of nonbilayer lipid phases in vivo. Proc. Natl. Acad. Sci. USA87:4112-4114
830 O.Vaarala 28. Nurminen, M., M. Leinonen, P. Saikku, and P. H. Miikeli. 1983. The genus-specific antigen of Chlamydia: resemblance to the polysaccharide of enteric bacteria. Science 220: 1279-1281 29. Caldwell, H. D. and P. J. Hitchcock. 1984. Monoclonal antibody against a genus-specific antigen of Chlamydia species: location of the epitope on chlamydial lipopolysaccharide. Infect. Immun. 2: 306-314 30. Wicken, A. J., J. W. Gibbens, and K. W. Knox. 1972. Anti-teichoic acid antibodies and non-treponemal serological tests for syphilis. Infect. Immun. 6: 982-984 31. McNeil, H. P., J. E. Hunt, and S. A. Krilis. 1990. New aspects of anticardiolipin antibodies. Clin. Exp. Rheumatol. 8: 525-527
