THE Clq BINDING ASSAY IN SYSTEMIC LUPUS ERYTHEMATOSUS

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1 1282 THE Clq BINDING ASSAY IN SYSTEMIC LUPUS ERYTHEMATOSUS Discordance with Disease Activity ROBERT D. INMAN, J. K. K. FONG, B. A. PUSSELL, P. J. RYAN, and GRAHAM R. V. HUGHES To investigate the relationship of Clq binding assay (ClqBA) to disease activity in systemic lupus erythematosus (SLE), a retrospective study was carried out on 232 ClqBA performed in 33 patients with SLE. When initial values only were assessed (33 tests in 33 patients), there was no relationship between positive and negative ClqBA and abnormal renal function (P = 0.482, Fisher exact test). Of 87 tests performed during active renal disease, 34 (39%) were positive; of 48 tests during active non-renal disease, 25 (52%) were positive; of 83 tests when the disease was inactive, 45 (54%) were positive; and of 14 tests during episodes of infection, 10 (71%) were positive. Corresponding means for the ClqBA were as follows: renal 65.66, non-renal78.02, inactive 56.04, infection (no significant diarerences by Student s f-test). There was no significant relationship with the ClqBA when comparing active disease (renal and non-renal) with inactive disease ($ Yates = 1.875, P = 0.171). When renal function abnormalities were analyzed separately, the ClqBA values were inde- From the Division of Rheumatic Diseases, Hospital for Special Surgery, the Department of Medicine, Cornell University Medical College, New York, NY, and the Division of Rheumatic Diseases, Royal Postgraduate Medical College, London, England. Robert D. Inman, MD: Assistant Professor of Medicine, Corncll University Medical College, and Postdoctoral Fellow of the Arthritis Foundation;.J. K. K. Fong, MD: Research Fellow, Royal Postgraduate Medical College; B. A. Pussell, MD: Research Fellow, Royal Postgraduate Medical College; Peter J. Ryan, MD: Research Fellow, Royal Postgraduate Medical College; Graham R. V. Hughes, MD: Head, Division of Rheumatic Diseases, Royal Postgraduate Medical College. Address reprint requests to Robert D. Inman, MD, The Hospital for Special Surgery, 535 East 70th Street, New York, NY Submitted for publication March 10, 1980 accepted in revised form July 25, pendent of azotemia or proteinuria ($ Yates = 1399, P = 0.237). Patients were seen with progressive renal disease who had consistently negative ClqBA, as well as patients with benign clinical courses despite elevated CIqBA. The discordance of the ClqBA results with disease activity in SLE highlights the limitations of immune complex (IC) determinations by a single technique, and stresses the importance of evaluating such tests in terms of both their specificity and sensitivity. These data further suggest that the relationship of IC to the disease process in SLE may be a complex one. Systemic lupus erythematosus (SLE) has been described as the prototype of immune complex disease in humans (1). Evidence for the role of immune complexes (IC) derives from analogy with experimental IC disease (2), data on complement consumption (3), immunofluorescent study of tissues (4), and more recently from demonstration of circulating IC. The Clq binding assay (ClqBA) as described by Zubler et a1 (5) successfully discriminated between normal controls and a group of SLE patients in a comparative study of immune complex assays by a World Health Organization (WHO) collaborative group (6). The ClqBA is reported to be positive in 6&91% of SLE patients (5,7), but the clinical usefulness of the ClqBA in a given patient remains ill-defined. In routine use of the test in SLE patients at the Hammersmith Hospital in recent years, it has become apparent that the presence of circulating C 1 q binding material does not necessarily correlate with the presence of renal disease or even with disease activity. In an attempt to clarify this issue, a retrospective review was carried out on the ClqBA in SLE and its relation to clinical disease activity. Arthritis and Rheumatism, Vol. 23, No. 11 (November 1980)

2 Clq BINDING ASSAY IN SLE 1283 PATIENTS AND METHODS The clinical records, serologic tests, and ClqBA results in 70 SLE patients attending the Hammersmith Hospital were reviewed. The following criteria had to be met for inclusion in the study: 1) at least four American Rheumatism Association criteria for the classification of SLE (8), together with at least one elevated DNA binding value, as measured by the Farr ammonium sulfate precipitation technique (9); 2) serum immune complex measurement by ClqBA; 3) sufficient clinical information at the time of each ClqBA to assess disease activity. Of the patients' charts reviewed, 33 fulfilled these criteria. Two hundred and eighteen (94%) of these tests were done when patients were receiving prednisone therapy; 94 (40%) while patients were on both azathioprine and prednisone. Clq was purified from fresh normal serum by the method of Yonemasu and Stroud (10) as modified by Pussell (7). Purity of the preparation was shown by a single line on double immunodiffusion against anti-whole human serum and by a single peak on sucrose density gradient ultracentrifugation. Radioiodination was carried out by the lactoperoxidase technique (11). Functional activity of the 1251-Clq after iodination was shown in a hemolytic assay by the use of serum specifically depleted of C lq by a solid immunoabsorbent. The "'I-Clq was stored in portions in liquid nitrogen until used. The ClqBA was performed by the method of Zubler (5) as modified by Pussell et a1 (7). Human IgG was purified from myeloma serum by ammonium sulfate precipitation and ion exchange chromatography. Aggregation was induced by heating at 63 C for 30 minutes. Insoluble aggregates were removed by centrifugation (1,500g for 30 minutes), and aggregated IgG was separated by gel filtration on Sephadex G-200 (Pharmacia). The aggregated IgG was added in specific quantities to normal human serum (NHS), incubated at 37OC for 30 minutes, and stored in aliquots in liquid nitrogen. For each assay a set of dilutions of the aggregates in NHS was thawed and used to determine a dose-response curve. A positive result was dehed as binding greater than 40 pg equivalent of aggregated IgG. The following clinical classification was used: Active renal disease was defined as an elevated serum creatinine (> 125 pmol/liter) or a 24-hour urinary protein greater than 1 gm. Causes of renal dysfunction other than lupus nephritis, such as infection, calculi, and drug side effects, were excluded. Any of the following were classified as active non-renal disease: arthritis, fever (culture negative), cutaneous vasculitis, pleuritis, pericarditis, documented neuropsychiatric abnormalities, thrombocytopenia, hemolytic anemia, and alopecia. Table 1. Clq binding assay in sera of patients with SLE No. Clq binding No. assays positive (%) Active renal disease (39%) Active non-renal disease (52%) Inactive disease (54%) Infection (71%) Total 232 I14 (49%) u I 6ob mo Renal Non-renal Jnact1ve S@lC Q ~ Figure 1. Distribution of ClqBA results in clinical subsets of patients with SLE. (From 232 tests in 33 patients.) Not classified as evidence of active disease were the following: arthralgia without synovitis, headache without objective findings, mood changes without neurologic findings or thought disorder, aseptic necrosis of bone, and fever attributable to documented infection. Episodes of documented infection were analyzed separately. An assessment was performed of the clinical status of the patient on the date that the ClqBA was taken, then assignment to the appropriate clinical subset was made. This assignment was done without knowledge of the ClqBA result of that date. For individual patients, the most common transitions during the course of the disease were between asymptomatic, inactive disease and active non-renal disease. In general, renal dysfunction persisted once it had been documented. In 3 patients with renal disease, episodes of infection occurred with corresponding assignment to the category of infection during that period. RESULTS When only initial values were assessed (33 tests in 33 patients), there was no relationship between positive ClqBA and abnormal renal function (P = 0.482, Fisher exact test). Of 87 tests performed during renal disease, 34 (39%) were positive; of 48 tests during active non-renal disease, 25 (52%) were positive; of 83 tests when the disease was inactive, 45 (54%) were positive; of 14 tests during episodes of infection, 10 (71%) were positive (Table 1). Corresponding mean values (+ standard error) for the ClqBA were as follows: renal (+ 13.2); non-renal ( ); inactive (+ 7.78); infection ( ). There was no significant difference between these mean values by Student's t-test. The distribution of test values in each subset (Figure 1) revealed that negative ClqBA was the most common result, with the exception of the infection group. ClqBA performed at the time of urgent admission to the hospital were analyzed (Figure 2). In patients admitted with non-renal disease, 5 of 16 (33%) had nega-

3 ~~ 1284 INMAN ET AL 500 -v , 0... : tive results of tests. In patients admitted with active renal disease, 4 of 16 (25%) presented with negative test results. By Student's t-test there was no significant difference between the Clq values in these admission tests in renal and non-renal patients.* The number of positive and negative test results during active SLE (renal and non-renal combined) was compared with inactive disease. During active disease, 135 assays were performed, 59 of which were positive. During disease inactivity, 45 of 83 tests were positive. Chi square analysis showed the results of ClqBA to be independent of disease activity (2 Yates = 1.875, P = 0.171). When renal function abnormalities were analyzed separately, the C lqba results were independent of azotemia or proteinuria, as seen in Table 2 (2 Yates = 1.399, P = 0.237). Figure 3 illustrates the course of a patient with progressive renal insufficiency and negative ClqBA. There was no significant correlation of ClqBA with a-dna binding (r = 0.286) or with CH50 (r = 0.048). DISCUSSION This retrospective study revealed that 61% of serum samples in patients with lupus nephritis had no immune complexes detectable by C 1qBA. Conversely, 54% of the sera of SLE patients who were clinically well had elevated IC levels by the ClqBA. Changing degrees of proteinuria or azotemia correlated poorly with *Differences between the values at the time of urgent admission and those during disease inactivity were not statistically significant changing levels of IC. These results are at variance with some previous reports of ClqBA in SLE patients. Nydegger observed general concordante between '"I-C lq precipitation and disease activity. This was based on 4 patients, however, without a defined selection process (3). A more recent study (12) reporting a positive correlation of Clq and a graded scale of disease activity was based on single point assessment in each patient, and details of clinical activity, including renal involvement, were not stated. Immune complex measurement in SLE by other assays have had variable results. Concordance of disease activity and IC levels has been reported for the Clq deviation test (13) and the polyclonal latex agglutination inhibition test (14), while the lymphocyte rosette inhibition assay correlated poorly with activity (15). The Raji cell assay and the monoclonal rheumatoid factor assay (16) showed concordance with nonrenal disease but not with renal disease. The possibility exists that these findings represent a subset of SLE patients. Although inclusion criteria stipulated at least one elevated antidna value at some point, this existed in almost our entire SLE population. In our review, no SLE patient had to be excluded because of a continually normal DNA binding. The analysis was based only on patients in whom the ClqBA was performed. Because the use of this assay may vary among physicians, a potential bias enters the data for this reason. It is known that different preparations of heataggregated IgG will yield different results when several laboratories are compared (6). The proposal of the WHO to establish standard reference preparations will provide valuable comparative data between different research laboratories. The collaborative studies forthcoming should resolve some of the discrepant data presently in the literature on circulating immune complexes. Our findings were that of the 33 patients, 24 (72%) had an elevated ClqBA at some time, which is comparable to the 18 of 23 patients (78%) reported by the group in Geneva (6). Table 2. ClqBA in relation to renal disease in patients with SLE (2 = 1.399, not significant at P c 0.1) ClqBA ClqBA static elevated negative Renal function* or risingt or fallingt Static abnormal or worsening Normal or improving 8 15 * Serum creatinine change 210% of previous value or 24-hour urinary protein change 21 gm compared with previous value. t Change 22Wo of previous value for ClqBA.

4 Clq BINDING ASSAY IN SLE i, < % V I I I I I I 1 1 I I I I I 1 May 1978 Nov Jan 1979 Figure 3. Patient with progressive renal insufficiency with negative ClqBA. Normal serum creatinine less than 125 pol/liter. Several factors may account for the discordance of IC levels by ClqBA and disease activity found in this study. Intravascular complexes may not be the nephritogenic agents. In membranoproliferative glomerulonephritis, the immunoglobulin content of serum Clq reactive materials differs from that deposited in the glomeruli, suggesting that circulating and deposited complexes may be quite distinct (17). Since DNA alone binds effectively to glomerular basement membrane (1 8), glomerular injury may result from such complexes formed in situ, rather than by the trapping of circulating IC. Pathogenic IC may not be detected by a given assay. Size factors relate to the pathogenicity of complexes, and different assays detect IC over variable size ranges. ClqBA may not be detecting the truly pathogenic IC if these complexes do not fix the classical pathway of complement. Conversely, positive results in the ClqBA might result from other substances reactive with Clq, such as DNA, bacterial lipopolysaccharide, collagen, certain amino acids, and pyridoxal-5 -phosphate (1 9). The assay of IC measures a static point in a dynamic process of persistent antigenemia, sustained antibody formation, and clearance by the reticuloendothelial system. If any factor in this balance, such as release of antigen, is episodic rather than in a steady state, then the assay may vary as antigen:antibody ratios change with time. The discordance of IC levels and renal disease in SLE found in this study emphasizes that the dynamics of IC deposition in the kidney is a complex sequence of events. Glomerular localization may depend on the avidity of the antidna antibodies (20) and on interaction with glomerular complement receptors (21,22). Both vascular volume (23) and preexisting glomerular damage (24) may be determinants of IC uptake. Intrarenal deposition of localized IC appears to be a function of the phagocytic mononuclear cells in the mesangium (25). In the clinical management of SLE, it is now known that patients may remain clinically well even with antidna antibody titers that are persistently raised for long periods (26). It is also evident that elevated ClqBA values should not be interpreted in isolation as evidence of active disease or renal involvement in SLE. ACKNOWLEDGMENTS Gratitude is expressed to Ms Hjordis Kerezman for secretarial assistance and to Dr. Robert P. Kimberly for assistance in statistical analyses.

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