Complement activation in patients with systemic lupus erythematosus without nephritis

T. E. Mollnes, H.-J. Haga1, J. G. Brun2, E. W. Nielsen3, A. Sjöholm4, G. Sturfeldt5, U. Mårtensson4, K. Bergh6 and O. P. Rekvig7

Department of Immunology and Transfusion Medicine and
3 Department of Anaesthesiology, Nordland Central Hospital, Bodø,
1 Department of Rheumatology and
7 Institute of Medical Biology, University of Tromsø,
2 Department of Rheumatology, Haukeland University Hospital, Bergen,
6 Department of Microbiology, University of Trondheim, Norway and
4 Department of Medical Microbiology and
5 Department of Rheumatology, University of Lund, Sweden

Correspondence to: T. E. Mollnes, Department of Immunology and Transfusion Medicine, Nordland Central Hospital, N-8092 Bodø, Norway.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To study the association between disease activity and complement activation prospectively in patients with systemic lupus erythematosus (SLE).

Patients and methods. Twenty-one SLE patients were examined monthly for 1 yr. Disease activity, autoantibodies, conventional complement tests and the following complement activation products were investigated: C1rs–C1inh complexes, C4bc, Bb, C3a, C3bc, C5a and the terminal SC5b–9 complement complex (TCC).

Results. Modest variation in disease activity was noted. None of the patients had nephritis. Flare was observed at 27 visits. Four patients had anti-C1q antibodies in conjunction with modestly low C1q concentrations. The complement parameters were rather constant during the observation period. Slightly to moderately decreased C4 (0.05–0.10 g/l) was found in 10 patients and severely decreased C4 (<0.05 g/l) in seven patients. Decreased C4 was not associated with increased complement activation. Complement activation products were either normal or slightly elevated. TCC was the only activation product correlating significantly with score for disease activity at flare. None of the variables tested predicted flares.

Conclusion. Complement tests are of limited importance in routine examination of SLE without nephritis, although TCC is suggested to be one of the most sensitive markers for disease activity.

KEY WORDS: Systemic lupus erythematosus, Complement, Terminal complement complex.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Various laboratory tests including antibodies to native DNA and C1q, and different complement tests, have been used in the evaluation of systemic lupus erythematosus (SLE) patients to supplement clinical examination in order to optimize treatment and to consider prognosis [1, 2]. The complement system has long been known to be activated in exacerbations of SLE, particularly reflecting nephritic activity. It has been debated whether this complement activation is important in the pathogenesis of SLE or whether it is an innocent epiphenomenon. The association between complement deficiencies and SLE supports an important role for complement in preventing immune complex-mediated tissue damage [3, 4]. However, increased activation of complement without deficiencies may also contribute to the tissue damage in SLE, since it was recently shown in an experimental mouse SLE model that specific inhibition of complement abolished the development of the disease [5].

Three approaches are used to evaluate the complement system in vivo. Haemolytic assays, including total haemolytic activity (CH50) for the classical and alternative pathway, are mostly used for assessment of whole complement function. Individual components can also be measured functionally with haemolytic tests. Antigen concentrations of individual components, e.g. C3 and C4, reflect the level of the circulating components irrespective of their functional state. Finally, various complement activation products are measured as indicators of complement activation using novel enzyme immunoassays (EIAs) with high specificity and sensitivity.

The use of complement analyses for the assessment of disease activity in SLE has recently been reviewed [6]. The literature on complement and SLE is conflicting for several reasons: the patient populations have been heterogeneous with respect to disease activity and organ involvement; different complement tests have been included in the various studies; and only a very few of the ~30 studies published have been prospective with analyses at predetermined intervals [7, 8].

We have established a broad spectrum of assays for complement activation products in our laboratory based on monoclonal antibodies (mAbs) reacting with neoepitopes exposed in the activation products [9]. Seven of the most representative activation products were selected for the present study, covering both the classical pathway (C1rs–C1inh complexes and C4bc), the alternative pathway (Bb), C3 (C3a and C3bc) and the terminal pathway [C5a and the terminal SC5b–9 complement complex (TCC)] (Fig. 1Go). The aim of the study was to examine the role of these and conventional complement tests in the evaluation of disease activity in SLE.



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FIG. 1.  A schematic illustration of the complement system. The activation products measured in the present study are indicated in boxes. C1rs–C1inh, the complex formed between C1r, C1s and C1 inhibitor when C1 is activated, is the very first activation product of the classical pathway. C1 activates C4 and C2, and the C4bc activation products (C4b, iC4b and C4c) indicate further activation of the classical pathway. Bb is an activation product of the alternative pathway factor B. Activation of C3 was detected by measuring the anaphylatoxin C3a and the activation products C3bc (C3b, iC3b and C3c). The anaphylatoxin C5a indicated activation of C5. TCC (terminal C5b–9 complement complex), composed of all the terminal components, demonstrates activation of the whole cascade to its very end.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inclusion of patients
Twenty-one patients (20 females, one male) fulfilling four or more of the American Rheumatism Association (ARA) classification criteria for SLE [10] were included in this prospective study. The patients were examined once monthly for 1 yr by one physician (HJH) at one centre. There were a total of 228 visits with an average 11 visits per patient (range 2–16). Disease activity was assessed at each visit according to the doctor's global assessment (range 0–10) and the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) [11], where anti-DNA and complement were excluded as criteria since they were studied separately. SLEDAI assessed the disease activity during the last 10 days before the examination, while in the doctor's global assessment the disease activity since the last examination ~4 weeks before was assessed. Flare was defined as an increase in SLEDAI score of >=3 [12]. Specific organ damage was assessed by the SLICC (Systemic Lupus International Collaborating Clinics) damage index, which is a list of items considered to reflect damage in SLE regardless of its cause [13]. Written approved consent was obtained from all patients before inclusion. The study was approved by the local ethical committee.

Patient characteristics
Since the present study concerned unselected SLE patients investigated at one centre and retrieved from one area, most of the patients had mild disease [14, 15]. Thus, none of the patients had evidence of renal involvement, although five of the patients previously had evidence of nephritis. They were, however, in full remission during the study. Some selection of patients may be due to long travelling distances in the northern part of Norway. In addition, the more severely affected patients may not have accepted being included in this study. Lack of nephritis in this study is not due to the treatment of these patients at the department of nephrology, since these patients are also examined by the rheumatologists. The mean age at entry to the study was 46.7 yr (range 23–75). Mean duration from first SLE symptom was 19 yr (range 8–36).

Most of the patients were using one or more of the following drugs: non-steroidal anti-inflammatory drugs, prednisolone, antimalarials, azathioprine, and two patients were treated with monthly pulses (2 and 6 months, respectively) of 500 mg of cyclophosphamide due to cerebral vasculitis and progressive fibrosis of the lungs. During the study, the treatment was repeatedly adjusted according to disease activity. Three patients received both prednisolone and azathioprine, nine prednisolone only and four azathioprine only. The median dose of prednisolone was 10 mg/day (range 7.5–20) and that of azathioprine 100 mg/day (range 100–150). After her second visit, one patient died due to a cerebral haemorrhage.

Laboratory analyses
Blood samples.
Blood samples were collected at each visit. Analyses performed in the routine laboratory were: haemoglobin (Hgb), white blood cell counts (WBC), lymphocytes, neutrophils, platelets, erythrocyte sedimentation rate (ESR), creatinine, C-reactive protein (CRP), antinuclear antibodies (ANA; Hep-2 cell immunofluorescence technique) and anticardiolipin antibodies (IgG and IgM class; enzyme immunoassay). Anti-native DNA antibodies were measured using a `plasmid ELISA' as described previously [16]. There was a strong correlation, although not complete, between this assay and previously published results on the same sera using a calf thymus anti-double-stranded (ds) DNA ELISA [17].

Anti-C1q antibodies.
All samples were examined for anti-C1q antibodies using two different enzyme immunoassay techniques. The `total anti-C1q' assay uses intact C1q for coating and antibody binding under high ionic strength conditions [18] and the `anti-C1qCLR' assay uses purified collagen-like C1q fragments for coating and antibody binding under physiological ionic strength conditions [19]. Values were given in arbitrary units using selected patient sera containing high antibody concentrations according to both assays for the construction of calibration curves. One sample from each patient was examined for anti-C1q antibodies by immunoblot analysis [19]. One patient tested positively and the rest of the samples from this patient were therefore also included in this assay.

Complement tests.
Blood samples were obtained in empty vacutainer tubes for the preparation of serum or in ethylenediamine tetraacetic acid (EDTA) tubes for the preparation of EDTA plasma. The EDTA tubes were immediately cooled on crushed ice, and centrifuged at 2000 g for 10 min; plasma was aliquoted and stored at -70°C until analysed in one batch. The EDTA plasma samples were used for analyses of complement activation products, and C1q, C3 and C4. Serum was obtained by allowing the blood to clot at room temperature for 2 h and the tube was then centrifuged. Serum was removed, aliquoted and stored at -70°C until analysed in one batch. Serum was used for analyses of autoantibodies and for the functional complement analyses.

Complement haemolytic activity.
Classical and alternative CH50 were measured using microwell techniques as described in detail previously [20].

C1q.
The antigen concentration of C1q was measured using an EIA designed in our laboratory. Briefly, goat antiserum to C1q (Quidel, San Diego, CA, USA) was used as capture antibody diluted 1/10 000. Normal human serum was used as reference, defining 100%. A mAb to C1q (Quidel) was used as secondary antibody in the concentration 0.1 mg/l. The rest of the assay was performed as described previously for the general complement EIAs [21].

C1 inhibitor.
The antigen concentration of C1 inhibitor was measured by single radial immunodiffusion (NOR-Partigen, Behringwerke A/G, Marburg, Germany) and performed according to the manufacturer's instruction. C1 inhibitor function was measured using a chromogene substrate assay described in detail previously [22].

C3.
C3 was quantified using a double-antibody EIA. The mAb 13F6 reacting with C3c was produced in our own laboratory and used as capture antibody. Polyclonal anti-C3 (Behringwerke A/G, Marburg, Germany) diluted 1:10 000 was used as detection antibody. Enzyme conjugate was peroxidase-linked anti-rabbit Ig (Amersham International, Little Chalfont, UK) diluted 1:2000. The standard was a normal human serum pool with a known concentration of C3. Plasma samples were diluted 1:20 000.

C4.
C4 was quantified using a double-antibody EIA similar to the C3 assay. Polyclonal anti-C4 antibody (Quidel, San Diego, CA, USA) diluted 1:50 000 was used as capture antibody and the same antibody biotinylated as detection antibody. Enzyme conjugate was peroxidase-linked streptavidin (Amersham) diluted 1:1000. Plasma samples and standard were as described for C3.

Complement activation products.
Activation products from the classical pathway (C1rs–C1 inhibitor complexes and C4bc), the alternative pathway (Bb) and the final common pathway (C3a, C3bc, C5a and TCC) were all quantified using mAbs to neoepitopes exposed in the activation products, but hidden in the native components [9]. The analyses were performed with EIAs according to principles described in detail elsewhere [21]. Thus, the tests are described only briefly below. Most of the results are given in arbitrary units (AU)/ml, which are related to a standard of activated serum defined to contain 1000 AU/ml. The reason for giving results in arbitrary units is that many of the complexes and activation products are heterogeneous with respect to molecular weights and thus SI units cannot be calculated. The amount of neoepitope expression in these activation products, as quantified by the specific mAbs, is best given in AU referred to a standard with high amounts of these products (virtually complete in vitro activation of complement). For the classical pathway products, this standard was made by activating serum with heat-aggregated IgG and for the rest of the products standard was serum activated with zymosan. A description of procedures for the production of standards and the general performance of these assays is given in detail elsewhere [21].

C1rs–C1 inhibitor complexes (C1rs–C1inh).
These were measured using the KOK-12 mAb specific for a neoepitope in the C1 inhibitor when it is in complex with the protease. The assay is described in detail elsewhere [23]. Briefly, plates were coated with the KOK-12 antibody, reacted with plasma and control samples, and the complex detected using a cocktail of anti-C1r and anti-C1s antibodies.

C4bc (i.e. the sum of C4b, iC4b and C4c).
This was measured mainly as described in detail previously [24]. The mAbs to C1 inhibitor and C4bc were a kind gift from Professor C. E. Hack, Amsterdam, The Netherlands.

Bb.
The factor B activation product Bb was quantified using a commercial kit (Quidel) and performed according to the manufacturer's instruction.

C3a.
C3a was quantified as described [25] using mAbs kindly provided by PROGEN Biotechnik (Heidelberg, Germany).

C3bc (i.e. the sum of C3b, iC3b and C3c).
This was measured using the mAb bH6 specific for a neoepitope exposed in C3b, iC3b and C3c, and performed according to a procedure described in detail elsewhere [21].

C5a.
C5a was quantified by EIA as described previously using the neoepitope-specific mAb 4A2E10E2 as capture antibody [26].

TCC
. The terminal SC5b–9 complement complex was quantified using the neoepitope-specific mAb aE11 as capture antibody. aE11 recognizes an epitope exposed in C9 when C9 incorporates into the TCC complex. This epitope is not exposed in native C9. The assay has been described in detail previously [21].

Statistical methods
Statistical analysis was performed with SPSS/PC+ programs (SPSS Inc., Chicago, IL, USA) and aimed to correlate the biochemical data with disease activity at flares. Spearman's rank correlation coefficient was used to analyse associations between variables at flares. In patients with more than one flare, the mean value for each patient was used in the calculations. The Mann–Whitney test was used to compare groups. Prediction of flare was defined as the correlation between a variable at the examination before flare and the disease activity as measured by SLEDAI at the time of flare.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical activity
Renal involvement was not observed in any of the 21 patients on any occasion. The patients were treated according to clinical disease activity. Therefore, relatively minor variations in disease activity were noted during follow-up. This is illustrated by the modest variations in the disease activity parameters: doctor's global assessment ranged from 1 to 9 and SLEDAI ranged from 1 to 22 (median 4). The correlation between SLEDAI and doctor's global assessment was at the significance level (r=0.53; P=0.05). The mean value for ARA criteria was 5.81 (range 4–9). The SLICC damage index was 0.76 (range 0–3) and did not change significantly during the observation period.

Flare, defined as an increase in SLEDAI by >=3, was observed at 27 visits (11.8%) in 14 patients, who experienced 1–4 flares each. Most of the flares were modest and responded to treatment. Arthritis was found at 29 visits (12.7%) and infections at 18 visits. Most of the infections were minor and already under treatment: four herpes simplex, one herpes zoster, four influenza, two pneumonia, three urinary tract infections including one pyelonephritis, one laryngitis, one sinusitis and one oral candidiasis.

Routine laboratory analyses
Median and range values were Hgb 12.8 (8.3–15.1) g/dl, ESR 21 (2–94) mm/h, CRP<5 (<5–69) mg/l, WBC 5.4 (2.2–14.6)x109 /l and platelets 243 (129–592)x 109 /l. Creatinine was 67 (42–124) µmol/l and urinary analyses were normal except for the acute urinary tract infections reported above, supporting the clinical conclusion that none of the patients had nephritis.

Autoantibodies
Antinuclear antibodies, anti-native DNA and anticardiolipin antibodies.
Seventeen of the patients were ANA positive during the observation period, but all of the patients had previously been tested ANA positive. Six patients were anti-DNA positive during the whole observation period and another six had at least one sample during the period above the reference cut-off of 25 U, but all these were weak. There were no correlations between anti-DNA antibodies and complement activation products. Two patients had both IgG and IgM antibodies to cardiolipin, whereas one had IgG cardiolipin antibodies only.

Anti-C1q antibodies.
Four patients had detectable anti-C1q antibodies (Table 1Go). Two of these (patient A and B) were positive in both the `total anti-C1q' assay and the `anti-C1qCLR' assay and one (patient C) was positive only in the `anti-C1qCLR' assay. One (patient D) tested positive for anti-C1q in Western blot in all samples obtained during the observation period, but was negative in the other two assays. Patients A and C were positive for anticardiolipin antibodies, whereas only one of the 17 anti-C1q-negative patients had anticardiolipin antibodies. Patients A and B were positive for anti-DNA antibodies, one with high and one with slightly elevated levels, whereas patients C and D were anti-DNA negative. One of the patients with anti-C1q antibodies had arthritis and all of them had previously experienced arthritis, but none of them had ever had nephritis. The patients with anti-C1q antibodies did not have C4 levels significantly different from those without these antibodies. They all had elevated ESR, but only two of them had significant disease activity during the study.


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TABLE 1.  Relationship between anti-C1q antibodies and C1 concentration for the four patients testing positive for at least one anti-C1q antibody test. The 17 patients who tested negative for anti-C1q had a C1q concentration of 110% (94–118) (median and 95% confidence intervals) compared with 71% (44–86) for the four patients with anti-C1q antibodies (P = 0.006)
 
There was no correlation between clinical activity at flare, as evaluated by SLEDAI and doctor's global assessment, and the presence of antibodies to DNA, C1q or cardiolipin.

Complement tests
Haemolytic activity.
Total complement haemolytic activity was measured with assays specific for the classical (CH50-C) and alternative (CH50-A) pathway. The level of CH50 was relatively constant during the observation period in each individual patient. CH50-C was consistently low in six patients and normal in 10, whereas five had some values above and some below the lower reference limit. The variations over time were, however, also minimal in this latter group. CH50-A was in the normal range during the whole observation period in 19 patients, whereas two showed some values below the lower reference limit. Except for CH50-A and doctor's global assessment (r=-0.62, P=0.028), there was no correlation between haemolytic assays and clinical activity.

C1q.
The concentration of C1q was relatively constant during the observation period in each individual patient. Fifteen of the patients had median values >75% of the content in a normal human serum pool, whereas six patients had lower median values. Interestingly, all the four patients with anti-C1q antibodies were among these six (Table 1Go). The 17 patients who tested negative for anti-C1q had a C1q concentration of 110% (94–118) (median and 95% confidence intervals) compared with 71% (44–86) for the four patients with anti-C1q antibodies (P=0.006).

C1 inhibitor.
C1 inhibitor was evaluated by both antigen and function. All patients had fully normal values for both tests during the whole observation period. There was a close correlation between these two tests (r=0.74, P=0.002), as seen in normals. There was no correlation between clinical activity and either of these tests.

C3 and C4.
The concentration of C3 was rather constant and mostly normal during the observation period, and did not differ much between the individual patients. In contrast, C4 values differed more among the patients, but these were also remarkably constant during the observation period in each individual patient. Seven patients had median C4 values below 0.05 g/l, and 10 patients had values between 0.05 and 0.10 g/l (reference range 0.10–0.50 g/l). The median value for the whole population (all patients during the whole period of investigation) was 0.06 g/l. C4 was detectable in all patients, the lowest value being 0.02 g/l. The low C4 values were not associated with increased complement activation. Neither was there any significant correlation between the level of C4 and susceptibility to infection. Prednisolone, azathioprine or cyclophosphamide did not influence the C4 concentration. No correlation was found between C3 and C4 and SLEDAI, but C3 correlated with doctor's global assessment (r=-0.75, P=0.002), as did CH50-A, as mentioned above.

Complement activation products.
Activation of the classical, alternative and terminal pathways of complement was determined by quantification of the following activation products: C1rs–C1inh complexes, C4bc, Bb, C3bc, C5a and TCC. The levels of the various activation products were rather constant in each individual patient during the observation period and the differences between the patients were small. The degree of complement activation was very modest in the whole group when comparing patient values with normal reference values. Table 2Go shows the values for complement activation products during the whole observation period compared with levels observed at flares. Interestingly, TCC correlated significantly with SLEDAI at flare (r=0.72 and P=0.005). In contrast, no other variable tested in this study correlated with disease activity as measured at flares. Despite the correlation between TCC and clinical activity, the levels of TCC were largely below or around the upper reference limit (Table 2Go).


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TABLE 2.  Concentration of complement activation products in all patients during the whole observation period and during flares, compared to a reference range of normal healthy blood donors. Only TCC correlated significantly with clinical activity at flare (see the text)
 
Prediction of flares.
None of the variables tested were able to predict flares, defined as correlation between a variable at the examination before flare and disease activity as measured by SLEDAI at the time of flare.


    Discussion
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 Materials and methods
 Results
 Discussion
 References
 
The patients in the present study had relatively low disease activity and none had nephritis. Complement activation in SLE has been particularly associated with renal affection and our patient population may explain the modest degree of complement activation, as reflected by normal or slightly elevated activation products, and the lack of correlation between complement activation products and disease activity, except for TCC.

Four of our patients had anti-C1q antibodies by at least one of three methods. Such antibodies have been described in SLE and hypocomplementaemic urticarial vasculitis syndrome (HUVS) [19, 27]. Anti-C1q detected by Western blot is usually seen in patients with HUVS [19], but the patient in our study showing such antibodies had low disease activity and no signs of HUVS. Siegert et al. [28] reported that anti-C1q antibodies do not activate complement either in vivo or in vitro, probably due to the inhibitory effect of C1 inhibitor and C4b binding protein acting as potent regulatory proteins of the classical pathway. Consistently, in our study neither of these patients showed extensive complement activation. There was, however, a significantly lower C1q concentration in the patients with anti-C1q antibodies. The association between anti-C1q antibodies and low C1q concentrations could reflect the formation of C1q-containing complexes, thus leading to increased turnover of C1q [29]. We can, however, not exclude the possibility that anti-C1q antibodies may be of pathogenetic importance, although not associated with increased complement activation in the present study.

C1 inhibitor is occasionally low in SLE [30], or dysfunctional as described in seven of eight SLE patients [31]. These patients had no angioedema and genetic C1 inhibitor deficiencies or anti-C1 inhibitor antibodies were absent. We found completely normal levels for C1 inhibitor antigen and function in the present study, and suggest that the inconsistent finding for C1 inhibitor function may be of methodological origin. The very close correlation we found between C1 inhibitor antigen and function suggests that the function was preserved both in vivo and in vitro. If the samples are not obtained or stored properly, or if cold-dependent activation occurs in vitro, the functional activity will be reduced. A substantial discrepancy between the degree of in vivo and in vitro complement activation due to cold-dependent activation in an SLE patient has been described [32]. In order to perform reliable tests for functional complement activity and for activation products, it is critically important that the samples are handled according to strict guidelines [21]. Inappropriate handling of the samples may explain some of the many contradictory results published on complement activation products and SLE.

C4 concentrations were low during the whole observation period in half of the patients, but this was not associated with an increased complement activation, consistent with previous observations [33]. Low C4 levels may falsely be regarded as classical pathway activation. Several other factors may explain low C4 values in SLE. Partial defects or homozygous defects in either C4A or C4B will result in reduced levels of total C4. Reduced synthesis or increased catabolism of C4 without a corresponding complement activation may also explain low C4 values. Charlesworth et al. [34] found increased catabolism of both C3 and C4 in SLE, irrespective of disease activity. However, C4 was lower in active SLE, which was explained by a lower synthesis during periods with increased disease activity. Partial C4 defects were observed in several patients in the same study [34]. Synthesis, activation and increased catabolism may explain C4 fluctuations over time, in contrast to C4 defects per se. Swaak et al. [35] found a fall in C4, and thereafter in C1q and C3, in SLE patents with exacerbation of renal disease, in contrast to inconsistent complement findings in patients with extrarenal flares. In the case of an activation of C4, it has been speculated that an increased concentration of C4b binding protein may partly inhibit the cascade at this level and thus partially prevent activation of the rest of the cascade [36, 37]. In our patients, it is unlikely that the markedly lowered C4 values can be explained by complement activation alone, since the degree of C4 activation products was not correspondingly increased. Furthermore, there was no correlation between the use of prednisolone, azathioprine or cyclophosphamide and C4 concentration, indicating that these drugs did not reduce the synthesis of C4.

The most potent biological effects of complement result from activation of the terminal pathway (C5–C9). Inhibition at the level of C5 using a mAb binding to and blocking C5 cleavage alleviated the development of the disease in lupus-prone mice [5]. Inhibition of C5 implies that the biologically highly active products C5a and terminal C5b–9 complement complexes are not formed. In the fluid phase, C5a is difficult to detect due to its short half-life by binding to neutrophils. Soluble TCC is, however, easier to detect due to a longer half-life, and is a more reliable and stable indicator of complement activation in vivo than most of the other activation products [38]. A correlation between TCC and anti-C1q antibodies was recently demonstrated in SLE patients [39]. In the present study, TCC was found to be the only complement activation product correlating with disease activity at flare. None of the previous studies on SLE and complement activation have included such a complete panel of complement activation products as we did here. Thus, we suggest that even in patients showing low clinical activity, the complement system was slightly activated during flares and that TCC is one of the most sensitive laboratory markers to detect a minor increase in activity. Previous studies have provided evidence for classical pathway activation at least in some patients with flares of mild disease [7, 8]. Formation of TCC partly reflects local complement activation and this could be pathogenetically significant in mild as well as in severe SLE.

To our knowledge, this is the first prospective study including TCC as a marker in SLE where samples were obtained according to accepted guidelines for sample collection and preservation. Six previous studies have examined TCC in SLE patients. Three of these studies used serum instead of EDTA-plasma and the results are, therefore, unreliable with respect to in vivo activation since most of the TCC detected in serum is a result of in vitro activation. The other three studies [4042] collected samples correctly. Porcel et al. [40] found TCC to be a better discriminator between active and inactive disease than C3a and C5a, whereas they found no difference for iC3b. Their study, however, was not prospective, but compared different SLE patient groups cross-sectionally. Similarly, Horigome et al. [41] and Auda et al. [42] found higher TCC values in SLE patients than in controls.

Our data suggest that complement tests in general are of limited value in routine examination of patients with mild SLE and that TCC seems to be one of the most sensitive laboratory markers of disease activity in SLE patients, even in the absence of nephritis.


    Acknowledgments
 
Excellent technical assistance was provided by Hilde Fure, Grethe Bergseth and Tone Reitstad. Financial support was provided by Gythfeldt's legacy, Norsk Revmatiker Forbund, the Swedish Medical Research Council (project nos 7921 and 9528), the Swedish National Association against Rheumatism, King Gustaf V's 80th Birthday Fund, and the Foundations of Alfred Österlund, Greta & Johan Kock, Åke Wiberg and Nanna Svartz.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 5 October 1998; revised version accepted 1 April 1999.