ANTICARDIOLIPIN ANTIBODIES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

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1 382 ANTICARDIOLIPIN ANTIBODIES IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS GUNNAR STURFELT, OLA NIVED, RENEE NORBERG, RIGMOR THORSTENSSON, and KATARINA KROOK We studied a group of 59 unselected patients with systemic lupus erythematosus (SLE); these patients were from a defined population who lived in southern Sweden. We found that serum concentrations of anticardiolipin antibodies were increased in 32 SLE patients (54.2%). No significant correlation between increased amounts of anticardiolipin antibodies and clinical symptoms, such as thrombocytopenia or thrombosis, was found. Serial serum samples from 28 patients (12 patients were from the epidemiologic cohort) were analyzed. Sixteen of these 28 patients (57.1%) had increased levels of anticardiolipin antibodies; in most cases, there was no variation in these values with regard to clinical disease Bares or treatment. Increased concentrations of anticardiolipin were observed in 4 patients with cerebral infarction. However, very high concentrations of anticardiolipin antibodies were observed in several patients with inactive SLE who had no history of thrombosis or thrombocytopenia. Our results underscore the importance of studying unselected patient groups when From the Department of Rheumatology, University Hospital, Lund, Sweden, and the Department of Immunology, the National Bacteriological Laboratory, Stockholm, Sweden. Supported by grants from the Medical Faculty, University of Lund, Lund, Sweden, Foreningen for Bistgnd h Vanfora i Skhe, Greta och Johan Kocks Stiftelse, Riksforbundet mot Reumatism, and Alfred Osterlunds Stiftelse. Gunnar Sturfelt, MD, PhD: Associate Professor, Department of Rheumatology; Ola Nived, MD, PhD: Associate Professor, Department of Rheumatology; Rente Norberg, MD, PhD: Professor, Department of Immunology; Rigmor Thorstensson, PhD: Department of Immunology; Katarina Krook: Laboratory Assistant, Department of Immunology. Address reprint requests to G. Sturfelt, MD, Department of Rheumatology, University Hospital, S Lund, Sweden. Submitted for publication January 13, 1986; accepted in revised form August 21, correlating laboratory data with clinical manifestations of disease. In patients with systemic lupus erythematosus (SLE) and related autoimmune disorders, circulating antibodies that react with negatively charged phospholipids (especially cardiolipin) have been shown to correlate with venous and arterial thromboses, thrombocytopenia, and recurrent fetal loss (1-3). The prevalence and the clinical importance of these antibodies have also been discussed in relation to the occurrence of human and mouse monoclonal antibodies against denatured DNA, and their cross-reactivity with cardiolipin and other phospholipids (4-6). We studied the prevalence of anticardiolipin antibodies and their association with clinical symptoms and disease in SLE patients. In this study, we analyzed sera from SLE patients who were part of an epidemiologic study that was conducted within the health care district of Lund and Orup, Sweden (7). Sera from consecutive patients with major SLE symptoms were also analyzed. PATIENTS AND METHODS Patients and serum samples. During 1980, a prospective study of SLE patients was started at the Department of Rheumatology, University Hospital, Lund. All patients with the diagnosis of SLE (8) who were living within the Lund-Orup health care district were included (7). An SLE clinic was then organized, and patients from a larger area of southern Sweden were also referred there for medical care. The patients were seen at regular intervals of &8 weeks, or at intervals that were appropriate to the course of the disease. Sera from 59 of 62 patients with SLE who lived within the Lund-Orup health care district in January 1983, Arthritis and Rheumatism, Vol. 30, No. 4 (April 1987)

2 ANTICARDIOLIPIN ANTIBODIES IN SLE 383 Table 1. Anticardiolipin antibodies in the epidemiologic cohort of systemic lupus erythematosus patients living in southern Sweden Anticardiolipin antibody* <3 AU 3-5 AU AU 210 AU IgG antibodies (n = 59) No. positive % positive IgM antibodies (n = 31)1 No. positive % positive * See Patients and Methods for details. t IgM antibodies were measured only in the sera which contained no detectable IgG anticardiolipin antibodies. were included in the present study. Detailed clinical data on these patients have been reported previously (8). We analyzed serum samples that had been taken during the first visit to the SLE clinic. Sera were also collected from 28 patients who had been sequentially followed up and who, during the time of observation (6-42 months, median 20 months), exhibited evidence of major symptoms of SLE, as previously defined (8). Twelve of these patients were included in the epidemiologic study (7). Fifteen were patients who lived outside the Lund-Orup district, but had been referred to the hospital; they were followed up in the same manner as the epidemiologic group. One additional patient, whose SLE was under control, was receiving treatment at the Department of Nephrology; serial serum samples from this patient were also studied. Blood was drawn at room temperature and at 37 C. Sera were stored at -70 C until analysis. Measurement of anticardiolipin antibodies. Antibodies against cardiolipin were determined essentially as described by Harris et al (1); however, we used an enzymelinked immunosorbent assay (ELISA) technique and alkaline phosphatase-conjugated monospecific anti-human y chain or anti-p chain antibodies (Orioq Diagnostica, Espoo, Finland). The monospecificity of the conjugated antibodies was established in the ELISA by testing reactions with immunoglobulins of defined isotypes other than the specific ones. Sera were diluted 1:60 and were tested on polyvinyl chloride microtiter plates. The enzyme reaction was measured as absorbance at 405 nm (Titertek Multiscan MC; Flow Laboratories, McLean, VA). Measurement of other antibodies. The lupus anticoagulant was detected by measurement of nonactivated and activated thromboplastio time, using a plasma recalcification system to measure anticoagulant activity, as previously described (9). All determinations of the lupus anticoagulants were made at the Department of Coagulation Disorders, Malmo General Hospital, Malmo, Sweden. Antinuclear antibodies, including antibodies to double-stranded DNA (dsdna), were determined by a routine immunofluorescence technique. Control subjects and serum samples. Sera (n = 156) were collected from voluntary blood donors and healthy laboratory personnel (age range years). These sera were used as controls. When establishing the level of cardiolipin antibodies in control sera, 5 control sera were randomly selected to be included in all experiments. The mean absorbance of these sera was compared with the absorbance of each of the other control sera. Only 4 of the 156 control sera gave an absorbance higher than 2 standard deviations above the mean in the IgG test. None had an absorbance of more than 5 standard deviations above the mean. The standard deviation of the mean IgM anticardiolipin value was higher than that of IgG, and individual control serum samples showed very high absorption values. The mean + 3 SD was chosen as the upper level of normal for both IgG and IgM anticardiolipin antibodies. This value corresponds to 3 arbitrary units (AU). In all experiments, at least 5 representative control sera and 1 positive serum (a gift from Dr. G. Hughes, Hammersmith Hospital, London, UK) were included in every microtiter plate assayed. A second group of control sera was made up of all serum samples (n = 96) that had been sent for serologic streptococcal analyses (National Bacteriological Laboratory) during 1 week in January of These sera were obtained from patients whose ages ranged from 14 to 76 years. Statistical analysis. The nonparametric chi-square test for 2 independent samples was used when comparing the clinical features of patients who had elevated levels of anticardiolipin antibodies with those of patients who had normal concentrations of anticardiolipin antibodies. The relationship between anti-dsdna titers and anticardiolipin titers was analyzed using Spearman s rank correlation test (10). RESULTS Twenty-eight (47.5%) of the 59 patients from the epidemiologic cohort had a serum level of IgG anticardiolipin antibodies >3 AU (Table 1). Eighteen patients (30.5%) had a value 25 AU, but only 5 patients (8.5%) had values that exceeded 10 AU. IgM anticardiolipin antibodies were demonstrated in 4 of the 31 sera that lacked IgG class anticardiolipin antibodies (Table 1). Blood sampling at room temperature and at 37 C showed no influence on the results. The clinical features, which included thrombo-

3 384 STURFELT ET AL Table 2. Comparison of clinical features in unselected systemic lupus erythematosus patients with normal and elevated levels of anticardiolipin Butterfly rash Discoid lupus Arthritis Serositis Renal disease Neurologic disease Thrombocytopenia* Venous thrombosis Pulmonary vasculitis/embolism % with elevated anticardiolipin levels (n = 32) * Two patients had only IgM anticardiolipin antibodies. % with normal anticardiolipin levels (n = 27) cytopenia, thrombophlebitis, and neurologic symptoms, of patients who had increased serum concentrations of anticardiolipin antibodies were not significantly different from those of patients who had normal concentrations (Table 2). One patient had a history of cerebral infarction and was anticardiolipin positive. Five female patients had very high concentrations (>lo AU) of IgG anticardiolipin antibodies. One of them had a history of episodic hemianopia, thrombophlebitis, and 3 spontaneous abortions, but her SLE was clinically inactive during the period of observation. Another patient had thromboghlebitis, butterfly rash, thrombocytopenia, and glomerulonephritis at disease onset, but during the study, the SLE was clinically inactive. The other 3 patients had shown no evidence of major neurologic symptoms, thromboembolic manifestations, or thrombocytopenia, and had not had any abortions since the onset of SLE symptoms (14-39 years). Three of the 5 patients with anticardiolipin antibodies > 10 AU exhibited a positive Wassermann reaction. No other patient had a positive Wassermann reaction. Two women had histories of multiple (23) abortions; 1 was anticardiolipin positive, and the other was anticardiolipin negative. In the epidemiologic group, no relationship could be demonstrated between the results of tests for anti-dsdna antibodies and the results of tests for anticardiolipin antibodies. At the initial visit to the clinic, only 11 patients had active disease. For this reason, anticardiolipin antibodies were also determined in serial samples from a group of 28 SLE patients. Twelve of 28 consecutively followed patients had consistently normal levels of IgG and IgM anticardiolipin antibodies, while in- creased values were demonstrated in 16 of them (57.1%). IgG antibodies were demontrated in 15 patients, whereas l patient had IgM antibodies without demonstrable IgG antibodies throughout the study period. In most of these patients, antibody concentrations were moderately increased. Anticardiolipin levels >10 AU at any sampling were found in only 7 patients. Throughout the study, anticardiolipin concentrations were fairly constant in most patients, and showed no variations with clinical disease flares or with institution of corticosteroids and immunosuppressive therapy (Figures 1A-C). In occasional patients, anticardiolipin antibodies were found during the qisease flare, but were not detected after the start of corticosteroid treatment (Figure ID). One of the patients underwent plasmapheresis because of active kidney disease (Figure 2). The anticardiolipin antibody level, which was 7 AU initially, was <3 AU after the sixth plasmapheresis. Twelve weeks after plasmapheresis was completed, he developed neurologic symptoms. Results of computed tomography scans were consistent with a diagnosis of cerebral thrombosis. Anticardiolipin antibodies were not demonstrated at that time, but during the following weeks, the values increased slowly and reached the original level (7 AU) 10 weeks after the acute thrombotic episode. Thus, in this case, cerebral infarction occurred at a time when circulating anticardiolipin antibodies were not demonstrated. Lupus anticoagulants were found in 5 of 7 anticardiolipin positive patients investigated. The clinical features in relation to anticardiolipin antibody levels in the serially studied patients are given in Table 3. We found that all 4 patients with verified occlusive arterial cerebral lesions were anticardiolipin positive. The neurologic manifestations among the 4 anticardiolipin negative patients were myelitis in 1 patient, epilepsy in 1 patient, and hemianopia and peripheral neuropathy in 2 patients. Three of 5 patients with peripheral thrombophlebitis had increased amounts of cardiolipin antibodies in their serum samples. Occlusive vascular lesions of the lung, evaluated by ventilation-perfusion scintigraphy (1 I), were found in 6 of the 28 patients. All 6 patients had dyspnea and thoracic pain, but chest radiographs were normal and there was no more probable explanation for the clinical symptoms. Only 2 of them were positive for anticardiolipin antibodies. The levels of IgG anticardiolipin antibodies in the sera of healthy blood donors varied within rather

4 ANTICARDIOLIPIN ANTIBODIES IN SLE 385 Cardiolipin antibody units 40 4 Cardioiipin antibody units 401 2o i I, months 1 m cyclophospharnldm Prednisolone I, months A + aralhioprlne B Cardlolipin antibody units 40 1 Cardiolipin 401 antibody units Prednisolone mg/d months Prednisolone mg/d C Figure 1. Anticardiolipin antibody levels, disease course, and steroid and immunosuppressive therapy in 4 patients with systemic lupus erythematosus (SLE). A, A 30-year-old woman with SLE of 10 years duration. During the observation period, she developed fever and gastrointestinal pain caused by aseptic salpingitis. Ten weeks later, she had acute cerebral symptoms, and she died within 3 weeks from a large central cerebral infarction. Her serum concentrations of anticardiolipin were moderately elevated throughout the study. B, An 18-year-old woman with SLE that presented as glomerulonephritis, thrombocytopenia, fever, and cerebral involvement. Cerebral infarction was verified by computed tomography and angiography. This patient constantly had very high serum concentrations of anticardiolipin antibodies. C, A 20-year-old man with SLE, who was observed during 3 years of mild skin and joint symptoms. During the observation period, he develuped a flare of serositis that was treated with corticosteroids. The anticardiolipin levels were constantly elevated. D, A 62-year-old woman with mild SLE. After radiation treatment subsequent to radical surgery for breast cancer, the patient developed arthritis, serositis, and later, cerebral infarction, which was verified by angiography and computed tomography. Her serum concentrations of anticardiolipin were moderately elevated until the start of treatment with high doses of corticosteroids. Prednisolone dosage is mdday. D restricted ranges. However, in 26 of 96 serum samples that were sent to the laboratory for streptococcal serology, anticardiolipin antibody levels 2 3 AU were found. Twelve of them had values 2 5 AU, and 6 sera showed amounts 210 AU. Only 1 of the patients with a serum concentration 210 AU had an increased anticardiolipin value when reevaluated 1 year after the first examination.

5 386 STURFELT ET AL DISCUSSION A main objective of the present investigation was to study the occurrence of anticardiolipin antibodies in sera from unselected patients with SLE. A prospective SLE project in southern Sweden, including all SLE cases from a defined population (7,8), provided the basis for the study. The frequency of IgG or IgM anticardiolipin antibodies in the epidemiologic cohort was 54.2%. With the ELISA technique, the presence of IgM rheumatoid factor influences the determination of IgM antibodies in sera containing IgG antibodies of the same specificity. Therefore, anticardiolipin antibodies of IgM class were analyzed only in sera with normal levels of IgG anticardiolipin antibodies. The differences in experimental conditions and sensitivity of the Wassermann reaction versus the ELISA, as used to measure anticardiolipin antibodies, explain the discrepancy between the results of the 2 methods. The prevalence of anticardiolipin antibodies in our subjects was somewhat lower than that noted by Hams et al(1), who found anticardiolipin antibodies in 40 of 65 SLE patients. Eighteen of the patients in that study had a history of 1 or more venous thromboses, and 15 of those patients had elevated levels of anticardiolipin antibody. Moreover, thrombocytopenia occurred more frequently among the patients with anticardiolipin antibodies. In our unselected SLE group, however, thrombotic manifestations and thrombocytopenia were not significantly more com / weeks Figure 2. Effects of plasmapheresis and subsequent cerebral infarction on serum concentrations of anticardiolipin antibodies in a 20-year-old man with systemic lupus erythematosus (SLE) manifested by skin, joint, kidney, and central nervous system symptoms. This patient experienced an SLE flare when anticardiolipin antibody levels were within normal limits. Table 3. Comparison of clinical features with the presence of anticardiolipin antibodies in 28 patients with active systemic lupus erythematosus who were followed serially Arthritis Gastrointestinal vasculitis Neurologic disease Occlusive arterial manifestations Other manifestations Nephritis Mucocutaneous syndromes Pulmonary vasculitis/embolism Serositis Thrombophlebitis Thromboc ytopenia Anticardiolipin Anticardiolipin positive negative (n = 16) (n = 12) mon in patients with anticardiolipin antibodies than in those without. The discrepancy between our results and those of Harris et a1 (l), especially in regard to the incidence of peripheral thrombophlebitis in relation to the presence of cardiolipin antibodies, may be explained by differences in the techniques used (particularly the antigen preparations) or differences in the populations studied. The former possibility cannot be addressed until structurally defined antigens are available. Regarding the SLE populations, Harris and coworkers studied a selected group. Our patients, who were from the epidemiologic cohort, were not selected and should therefore be more representative of the broad clinical spectrum of SLE. We obtained blood samples at the patient s first visit to the clinic, which could be during any stage of the disease. Only a few patients had clinically active disease at that time. Since a prospective study might more accurately reflect the relationship between anticardiolipin antibodies and disease manifestations, we studied consecutive sera from 28 patients in whom major clinical symptoms (8) occurred during the observation period (642 months). The overall prevalence of anticardiolipin antibodies among these patients (57.1%) was similar to that of the epidemiologic group. Also among these serially followed patients, no clear-cut correlation was found between thromboembolic manifestations and elevated concentrations of anticardiolipin antibodies. An interesting observation, however, was that occlusive cerebrovascular lesions were seen exclusively among patients who were anticardiolipin positive. No consistent pattern of anticardiolipin antibody levels in relation to disease activity and drug therapy was seen. The fre

6 ANTICARDIOLIPIN ANTIBODIES IN SLE 387 quent finding that anticardiolipin concentrations among sequentially followed SLE patients were fairly constant supports the relevance of analyzing cumulative clinical data and anticardiolipin levels. Results of this study suggest a relationship between anticardiolipins and occlusive arterial disease, which has been noted in previous reports (12), but do not confirm the proposed association between venous thrombosis and increased levels of anticardiolipin (1). Since platelets are important in the pathogenesis of arterial thrombosis (13), one possible effect of anticardiolipin antibodies could be their influence on platelet function (14). However, the present study clearly shows that high concentrations of anticardiolipin antibodies can occur in SLE patients who have no evidence of clinically active disease and no history of thrombosis or thrombocytopenia. Therefore, the predictive value of determinations of anticardiolipin antibodies is probably limited. No explanation can be readily offered for the low concentrations of anticardiolipin antibodies in many patients with occlusive vascular lesions of the lungs. However, the methods used to detect such abnormalities (mainly scintigraphy) do not differentiate various causes of disturbed circulation in the lungs (1 1). It is our experience that anticardiolipin antibodies are present in certain patients who experience recurrent abortions and concomitant infarction of the placenta. Very high anticardiolipin concentrations are often found in these patients, and most of them show permanently low concentrations of complement factor C4 (15). Anticardiolipin antibodies react in vitro with structures of intact lymphoid cells and platelets (15). It has been suggested that the removal of anticardiolipin antibodies, e.g., by immunosuppression or plasmapheresis, should be of therapeutic value. One of our patients had repeated plasmapheresis, and had a cerebral infarction 3 months after the last plasmapheresis. Anticardiolipin antibodies had not been demonstrated after plasmapheresis was completed, and the antibodies were not found until a month after the acute infarction. This anecdotal observation, however, may indicate that the removal of circulating antibodies and the absence of anticardiolipin antibody cannot prevent the development of thrombotic manifestations. Sera from laboratory personnel and normal blood donors comprised the control samples. Both sexes were represented, although there was a slight preponderance of female donors. These blood donors are, to some extent, selected because individuals with biologically false-positive VDRL or Wassermann reactions were not accepted as donors. Moreover, it can be anticipated that people might abstain from donating blood during temporary disease states, e.g., upper respiratory infections. There was a low prevalence of increased levels of anticardiolipin antibodies in the control subjects; this low frequency in normal subjects has also been reported by others (1). Information about the occurrence of anticardiolipin antibodies in patients with diseases other than SLE is lacking. Therefore, 96 randomly selected sera, which had been sent to the laboratory for streptococcal serology, were analyzed for the presence of anticardiolipin antibodies. Elevated levels of antibody were found in 27.1%. One year later, repeat investigations of sera from the 6 patients who had the highest titers showed that antibodies remained in only 1 serum. This sample was obtained from an older woman who had a chronic staphylococcal infection. The results of this study were not unexpected, especially since antibodies that react with phospholipids are regularly obtained by experimental stimulation with polyclonal activators of bacterial origin. However, the results underscore the importance of cautious interpretation of the diagnostic significance of anticardiolipin antibodies. REFERENCES 1. Harris EN, Gharavi AE, Boey ML, Pate1 BM, Mackworth-Young CG, Loizou SV, Hughes GRV: Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 11: , Boey ML, Colaco CB, Gharavi AE, Elkon KB, Loizou SV, Hughes GRV: Thrombosis in systemic lupus erythematosus: striking association with the presence of circulating lupus anticoagulant. Br Med J 287: , Hughes GRV: Thrombosis, abortion, cerebral disease and the lupus coagulant. Br Med J 287:108%1089, Colaco CB, Elkon KB: The lupus anticoagulant: a disease marker in antinuclear antibody negative lupus that is cross-reactive with autoantibodies to doublestranded DNA. Arthritis Rheum 28:67-74, Lafer EM, Rauch J, Andrzejewski CJ, Mudd D, Furie B, Schwartz RS, Stollar BD: Polyspecific monoclonal lupus autoantibodies reactive with both polynucleotides and phospholipids. J Exp Med 153: , Shoenfeld Y, Rauch J, Massicotte H, Datta SK, Andre- Schwartz J, Stollar BD, Schwartz RS: Polyspecificity of

7 STURFELT ET AL monoclonal lupus autoantibodies produced by humanhuman hybridomas. N Engl J Med 308:414420, Nived 0, Sturfelt G, Wollheim F: Systemic lupus erythematosus in an adult population in southern Sweden: incidence, prevalence and validity of ARA revised classification criteria. Br J Rheumatol 24: , Sturfelt G, Nived 0: Clinical inconsistency, benign course and normal employment rates in unselected systemic lupus erythematosus. Clin Exp Rheumatol 3~ , Dahlback B, Nilsson IM, Frohm B: Inhibition of platelet prothrombinase activity by a lupus anticoagulant. Blood 62: , Siegal S: Non-parametric statistics for the behavioral sciences. Tokyo, McGraw-Hill, Fazio F, Wollmer P: Clinical ventilation perfusion scintigraphy. Clin Physiol 1: , Hams EN, Gharavi AE, Asherson RA, Boey ML, Hughes GRV: Cerebral infarction in systemic lupus: association with anticardiolipin antibodies. Clin Exp Rheumatol 2:47-5 1, Nilsson IM: Thrombosis and Treatment of Thrombosis. London, John Wiley & Sons, 1974, p Glueck HI, Kant KS, Weiss MA, Pollack VE, Miller MA, Coots M: Thrombosis in systemic lupus erythematosus: relation to the presence of circulating anticoagulant. Arch Intern Med 145: , Norberg R, Thorstensson R, Krook K, Sturfelt G, Nived 0: Antibodies against phospholipids (cardiolipin) in SLE. Protides Biol Fluids 33: , 1985