Anti-C1q antibody as a marker of disease activity in systemic lupus erythematosus
The present study was conducted to examine the usefulness of anti-Clq antibody as a marker of disease activity in Indian patients with systemic lupus erythematosus (SLE). We standardized the assay for detection of IgG anti-Clq antibody using ELISA. The normal cut-off level was determined by testing 57 healthy, age and sex matched controls to be 53 units/ml (mean +/- 2 SD). Patients with SLE (97 females and 13 males) were studied and the following parameters were obtained on all: SLE disease activity index (SLEDAI), anti-Clq, anti-ds DNA and C3. Correlations were tested between these parameters using Spearman’s rank correlation coefficients. Anti-Clq was found positive in 66 (60%) patients while anti-ds DNA was found in 78 (71%). The positive predictive values of anti-Clq and anti-ds DNA for lupus nephritis were 59 and 61 per cent respectively. The titres of anti-C1q correlated positively with SLEDAI (P
Keywords Anti-C1q – lupus activity – lupus nephritis -systemic lupus erythematosus
Assessment of disease activity in systemic lupus erythematosus (SLE) can be quite difficult. Sometimes an underlying sepsis can mimick flare of disease. Laboratory markers such as anti-ds DNA, C3, C4, CH50 and urinary sediment abnormalities provide an objective assessment of lupus activity1-5. Since all these parameters have their drawbacks, the search for new serum markers continues6-9.
A few studies have examined the role of anti-Clq antibodies as a marker of lupus activity10-15. Anti– C1q antibodies seem to correlate with active renal SLE12,14,15. One study showed that anti-Clq antibodies correlated with the proliferative forms of lupus nephritis and the rise in titres of anti-C1q preceded the development of clinical activity of disease by 6 months with a positive predictive value of 50 per cent”. Another study reported a correlation between the ongoing production of IgG anti-C1q and proliferative lupus nephritis16. Anti-Clq antibody is not specific to SLE; in fact, it has been described in rheumatoid vasculitis, urticarial vasculitis, idiopathic membranoproliferative glomerulonephritis and IgA nephropathy17-21. There are no data on the role of anti-C1q antibody in SLE in the Indian population. The present study was planned to evaluate the role of anti-Clq antibody as a marker of lupus activity and to document its association, if any, with organ involvement.
Material & Methods
Consecutive SLE patients (97 females, 13 males) were recruited from the Rheumatology Clinic of our hospital between April 1995 and March 1998. All the patients conformed to the 1982 Revised American College of Rheumatology criteria for the classification of SLE21. The disease activity was expressed as a clinical score ‘SLEDAI’ according to the method described by Bombardier et al22 . The work-up of each patient included, besides detailed history and physical examination, the following laboratory investigations : ANA by indirect immunofluorescence 23, C3 by single radial immunodiffusion 24 , anti-ds DNA by ELISA25 and anti-Clq antibody by ELISA12. Important reagents for these assays included: anti- C3, C1q, anti-human IgG-biotin conjugate and streptavidin-HRP conjugate (Sigma, St. Louis, USA) and anti-human IgG-FITC conjugate (Dakopatts, Denmark). Nunc ELISA plates (Denmark) were used for the anti-Clq assay. Anti– ds DNA kit was obtained from Diamedix Corporation (Miami, Florida, USA). Anti-C1q antibody was also estimated in 57 age and sex matched, healthy controls to establish the normal range, Routine investigations such as complete blood counts, ESR, urine analysis, chest radiograph, ECG, liver function tests serum urea and creatinine were performed in all patients. Other investigations which included renal biopsy, echocardiography, ultrasonography, CT/MRI were carried out as and when indicated. Criteria for lupus nephritis included 24 h urinary protein excretion > 0.5g and urine microscopy showing cellular casts or 5 or more red blood cells per high power field. Consenting patients meeting these criteria underwent kidney biopsy. The study design was a cross-sectional one and, therefore, no follow up assessment was planned.
Statistical analysis: Spearman’s rank correlation coefficient was used to test the correlations between the various parameters of disease activity. Chi square test was used to test the differences between proportions.
A positive standard for anti-C1q antibody was obtained as a gift from Professor M. Daha from Leiden (the Netherlands) and a ‘Standard Curve’ was obtained by plotting the optical density values against different dilutions of the positive standard. This standard curve was utilized to obtain the titres of anti-C1q antibody in the test sera. The mean anti– C1q titre of 57 healthy controls was found to be 34.44 units/ml (SD = 9.33). A level of 2 SD above this mean i.e., 53 Units/ml was taken as the positive cutoff. The normal reference range for anti-ds DNA according to the manufacturer of the kit was
A total of 110 patients with SLE were included in the study. The median age was 29 yr (range 1362 yr). The median duration of disease was 26 months (range 142 yr). The mean SLEDAI score was 11.137.79 (range 0-42), mean anti-C1q titre 80 +/- 79.66 units/ml (range 0-580), mean anti-ds DNA titre 1120.22 +/- 1465.91 units/ml (range 50-9600) and mean.C3 level 78.58 +/- 39.33 mg/dl (range 19-205). The correlation matrix between the above 4 parameters using Spearman’s rank correlation coefficient is shown in Table 1. It can be seen that the titres of anti-C1q antibody correlate positively with SLEDAI and anti-ds DNA, and negatively with C3 levels.
Sixty six patients (60%) tested positive for anti– C1q and 78 (71%) for anti-ds DNA. Sixty (54.5%) patients had evidence of lupus nephritis. The positive predictive values of anti-C1q and anti-ds DNA for lupus nephritis were found to be 59 and 61 per cent respectively. Of the 60 patients with lupus nephritis, 39 underwent kidney biopsy and histology showed WHO class I lupus nephritis in 1, class II in 7, class III in 7, class IV in 18 and class V in 6 patients. Thus, 25 patients had proliferative type of lupus nephritis (WHO class III or IV). No correlation was found between anti-C1q antibody and proliferative lupus nephritis. Positivity for anti-ds DNA and anti– C1q was compared in the 54 patients with moderately severe disease activity (SLEDAI>10). Anti-ds DNA and anti-C1q positivity was found in 45 of 54 (83.3%) patients and 39 of 54 (72.2%) respectively. Of the 9 patients negative for anti-ds DNA, 5 tested positive for anti-C1q. On the other hand, II of the 15 patients negative for anti-C1q, tested positive for anti-ds DNA. Thus, the two autoantibodies together provided laboratory evidence of lupus activity in 50 of 54 patients i.e., 92.5 per cent of patients with moderately severe degree of lupus activity. In the remaining 4, low C3 was demonstrable in 2. Thus, anti-ds DNA, anti-C1q and low C3, in combination, were able to corroborate evidence of lupus activity in more than 95 per cent of patients with SLEDAI score above 10.
The correlation between anti-C1q positivity and organ involvement at the time of patient recruitment is given in Table II. Involvement of skin was present in 67, mucosa in 22, joints in 28, pleura/pericardium in 15, CNS in 6 and kidney in 60 patients. Anti-C1q positivity was not found to correlate with any organ involvement.
– The frequency of anti-C1q in our patients with SLE (60%) was found comparable to that reported by Siegert et al (56%) from the Netherlands”. Anti– C1q titre was positively correlated with SLEDAI which means that it can serve to quantify disease activity. There was also a positive correlation with anti-ds DNA and a negative correlation with serum C3. These characteristics point towards a possible role in the pathogenesis of SLE. Anti-C1q antibody may be playing a role in the pathogenesis of SLE by way of formation of circulating immune complexes (C1q-anti-C1q) with subsequent deposition in tissues, local formation of immune complexes with C1q already deposited in tissues, and by interference with the activation of C1 or the interaction of C1q with C1q receptor. It must be remembered, however, that the antibody is not specific to SLE 17-20. We did not find a correlation of anti-Clq with any particular organ involvement. This is in sharp contrast to the observation made by at least two groups that anti– C1q correlates with proliferative forms of lupus nephritis i.e., WHO Class III or IV14-16. Walport et al13 from London also did not find any correlation with proliferative lupus nephritis but their overall positivity of anti-C1q in SLE patients was only 23 per cent. Obviously more studies need to be done to ascertain the exact status of this new serum marker in SLE. We did not address the question of its ability to predict a future lupus flare in this study.
The fact that anti-C1q antibody was able to corroborate evidence of disease activity in 5 of 9 patients with clinically active lupus where anti-ds DNA was found negative, supports the clinical usefulness of this marker. As our results show, more than 95 per cent of patients with SLEDAI score of more than 10, test positive for one or more of these parameters anti-ds DNA, anti-C1q and C3. Thus, anti– C1q antibody may serve as an additional tool for rheumatologist to document lupus activity, particularly in anti-ds DNA negative patients.
The work was supported by an intramural grant from All India Institute of Medical Sciences, New Delhi. The help provided by the residents of the Department of Medicine is gratefully acknowledged.
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A. Kumar, R. Gupta, T. Varghese, R.M. Pande* , V.K. Singal & O.P. Garg
Departments of Medicine & *Biostatistics, All India Institute of Medical Sciences, New Delhi
Accepted December 3,1999
Reprint requests: Dr Ashok Kumar, Associate Professor of Medicine & Head, Clinical Immunology & Rheumatology Service Department of Medicine, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029
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