Links between complement abnormalities and systemic lupus erythematosus

Systemic Lupus Erythematosus/Series Editors: D. Isenberg and C. Gordon Complement is an important effector pathway of innate immunity. In this review the role of complement in the pathogenesis of systemic lupus erythematosus (SLE) is described. There are important associations between both inherited and acquired complement component deficiencies and SLE. These have focused attention on identifying the relevant physiological role of the classical pathway of complement activation that appears to protect against the development of SLE. There is also unequivocal evidence that major histocompatibility complex (MHC) genes play an important role in determining both disease susceptibility and phenotype in SLE and its subsets. Accordingly, the role of complement genes located in the class III region (C4A, C4B and C2) in the genetic susceptibility to SLE is reviewed. The utility of the measurement of complement levels in monitoring disease activity and the clinical significance of hypocomplementaemia are discussed. Homozygous C1q deficiency is the strongest genetic susceptibility factor for SLE that has been identified in humans, but, paradoxically, SLE causes C1q consumption and is commonly associated with anti‐C1q antibodies. The role of complement in the pathogenesis of lupus, with particular emphasis on the role of C1q, is discussed in order to explain this paradox. Homozygous hereditary deficiency of each of the classical pathway components (C1q, C1r, C1s, C4, and C2) is associated with an increased susceptibility to SLE [1]. Both the severity of disease and the strength of this association is greatest for homozygous C1q deficiency followed in turn by homozygous C4 and C2 deficiency. Thirty‐eight of the 41 patients with homozygous C1q deficiency reported to date have developed a clinical syndrome closely similar to SLE (recently reviewed in [2]). In the affected patients rash occurred in 36, glomerulonephritis in 16, and cerebral disease in seven. Antinuclear antibodies (ANA) were reported in 24 and antibodies to extractable nuclear antigens in 15 of these patients, but notably, the incidence of anti‐double‐stranded DNA antibodies was low, positive in only five of the 24 patients tested. Hereditary deficiency of C1s and C1r is rarer than that of C1q deficiency and, in the majority of cases, deficiencies of both components coincide [3–5]. In such cases C1r levels are usually absent with levels of C1s below 50% of normal. As in homozygous C1q deficiency, SLE occurs in the majority of these individuals (seven of the 11 reported cases). Selective C1s deficiency, i.e. with normal C1r levels, has been reported in association with SLE [6]. In homozygous C4 deficiency, the prevalence of SLE is approximately 75% and the lupus illness is of moderate severity [7]. Homozygous C2 deficiency is the commonest inherited classical pathway complement deficiency with an approximate prevalence in Western European Caucasoid populations of 1:20 000. In contrast to homozygous C1q deficiency, the majority of affected individuals are probably healthy. SLE may occur in up to 33%, although this figure may be an overestimate due to ascertainment artefact, and the severity of the illness is comparable to that seen in SLE patients without homozygous complement deficiency. In contrast to the classical pathway components, homozygous C3 deficiency predisposes to recurrent pyogenic infections and membranoproliferative glomerulonephritis (MPGN), but SLE is rare [8]. What do the clinical observations in individuals with homozygous classical pathway component deficiency teach us about the pathogenesis of SLE? They suggest that there is a physiological function of the classical pathway of complement activation that protects against the development of SLE. Furthermore, the hierarchy of susceptibility and the severity of lupus, according to the missing classical pathway protein (C1q > C4 >> C2), suggest that the early part of the classical pathway plays a key protective role against the disease. Since one of the major roles of the classical pathway is that of host defence against infectious disease, one hypothesis is that classical pathway component deficiency predisposes to SLE because of impaired resistance to an infectious trigger. There is little evidence in favour of this hypothesis. Classical pathway complement deficiencies are not associated with an overt increase in susceptibility to viral or fungal infections. Although complement deficiency is strongly associated with the development of pyogenic bacterial infections, such infectious agents are not strong candidates for the induction of SLE. Other immunodeficiencies associated with increased susceptibility to pyogenic bacterial infections are not associated strongly with the development of SLE. Complement plays a key role in the clearance and processing of immune complexes and cellular debris and the evidence will be summarized that it is the role of the classical pathway that protects against the development of SLE. In view of the link between homozygous complement deficiency and SLE, it was hypothesized that partial deficiencies of C4 or C2 might also increase disease susceptibility. C4 is the product of two genes, C4A and C4B, and the expressed C4A and C4B proteins have slightly different functional activities. There is good evidence that C4A null alleles are important in SLE. An increased prevalence of C4A null alleles in patients with SLE has been found in many different populations, independent of other MHC associations. These data are reviewed below. C4 is encoded by two tandemly duplicated genes, C4A and C4B, within the class III MHC region. Human C4A and C4B are highly polymorphic with variants including non‐expressed or null alleles for which no protein product is identifiable (expressed as C4AQ*0 and C4BQ*0, i.e. quantity zero). A single C4 null allele may be seen in up to 30% of healthy Caucasoid subjects with approximately 4% having homozygous C4A deficiency and 1% homozygous C4B deficiency. Different allotypes of C4A and C4B can be detected by differences in electrophoretic mobility [9], but phenotyping is difficult in patients with active disease in whom C4 levels are usually very low because of complement activation and consumption in vivo. Following the molecular cloning of the C4 genes [10], molecular typing mechanisms have enabled more accurate determination of C4 heterozygous and homozygous deficiency states. There is a strong association between C4AQ*0 null alleles and SLE in Caucasoid patients [11–25]. In some studies, the association shows a gene ‘dose‐dependent’ effect: relative risk estimates for SLE in the case of homozygous C4AQ*0 deficiency have been between 9.7 [25] and 16.86 [14], whilst estimates in heterozygous deficiency range from 2.3 [25] to 4.87 [22]. However, the major source of C4AQ*0 alleles is the commonest Caucasoid MHC haplotype: HLA‐A1, ‐B8, CW‐, ‐DR3, C4AQ*0, C4*B1, C2*C, B*fS. In view of this, it is extremely difficult to identify which is the relevant disease‐susceptibility gene within the MHC, since the particular allotypic variants of the many genes within the MHC are usually inherited en bloc, in what is termed an MHC haplotype. There are two ways around this problem. The first is to try to identify lupus patients bearing relatively unusual haplotypes which are the products of recombination at some point in human history. However, individuals bearing these recombined haplotypes are uncommon. In one study, the C4AQ*0 null allele frequency was determined in Caucasoid SLE patients who were HLA‐DR3 negative. Eighteen of 30 (60%) lupus patients had extended haplotypes containing a C4 null allele compared with 22 of 60 (37%) healthy Caucasoid controls (P < 0.05) [26]. The second approach is to analyse the HLA associations of SLE in ethnic groups in which other HLA haplotypes are seen, to test the hypothesis that the association with C4AQ*0 is present in different populations. A number of studies showed significant associations between C4AQ*0 deficiency and SLE without any consistent association with HLA‐DR3 [14, 15, 27–31]. For example, in African‐American patients the presence of a C4AQ*0 allele confers a relative risk of 4.5 despite no association with HLA‐DR3 [29]. Furthermore, in Japanese populations where the prevalence of HLA‐DR3 is extremely low, an association with C4AQ*0 was demonstrated [30]. However, there are also a significant number of studies which have not shown a significant association between SLE and the presence of C4AQ*0 alleles. In a study of French‐Canadian and non‐French‐Canadian lupus patients, a significant association with C4AQ*0 could be demonstrated only in the non‐French‐Canadian group [32]. Additionally, a Spanish [33], Greek [24] and a recent Caucasoid study [34] failed to detect a significant association between lupus and the presence of C4AQ*0 null alleles. It remains uncertain what is the relevant disease‐susceptibility gene or genes within the MHC. Other candidate genes within the MHC include those encoding (i) class II gene products, controlling the repertoire of peptide presentation to T cell receptors; (ii) components of the antigen processing machinery, e.g. HLA‐DM, ‐DO and proteins of the TAP (transporters associated with antigen processing) complex; (iii) the cytokines, tumour necrosis factor‐alpha and lymphotoxin and (iv) the many other proteins encoded in the MHC. Recent work has demonstrated that B cells and resting T cells from healthy HLA‐B8, ‐DR3 positive individuals express markedly reduced levels of Fas (CD95/APO‐1) compared with cells from HLA‐B8, ‐DR3 negative matched controls [35]. This is of direct relevance to SLE where, as discussed below, inefficient clearance of apoptotic cells has been implicated in the disease pathogenesis. Evidence for an association between C4BQ*0 alleles and SLE is much less strong than for C4AQ*0 alleles. Indeed the majority of studies have found no increase in the C4BQ*0 allele frequency in Caucasoid [12, 14, 15, 21, 26, 34], Asian [15, 27, 31] and African‐American populations [14, 28, 34]. It is therefore necessary to explain why a partial deficiency of C4A and not C4B might predispose to SLE. The C4A isotype shows preferential binding to amino groups, forming amide bonds, and binds particularly to proteins, for example in immune complexes [36], whilst C4B is haemolytically more active than C4A, reflecting increased ester binding of C4B to carbohydrates on erythrocytes. It could be hypothesized that deficiency of C4A results in impaired processing of immune complexes or apoptotic cells which, as discussed below, may be important in the pathogenesis of SLE. Although one early study did detect an association between heterozygous C2 deficiency and SLE [37], later studies have not confirmed this [12, 18, 38, 39]. The C2 null allele (C2Q*0) occurs, in the majority of cases, in association with the haplotype HLA‐A25, B18, CW‐, DR2, C4A*4, C4B*2, Bf*S [40–42]. The molecular basis of C2 deficiency associated with this extended haplotype consists of a 28 base pair genomic deletion that causes skipping of exon 6 during RNA splicing, resulting in the generation of a premature stop codon [43]. The frequency of this extended haplotype was not raised in a study of 248 European lupus patients [38] and none of the HLA‐B18 positive lupus patients in another Caucasoid study possessed the complotype, C4A*4, CAB*2, Bf*S [12]. Furthermore, the frequency of the 28 base pair deletion associated with type I C2 deficiency was not significantly elevated in a study of 86 Swedish lupus patients [39]. Therefore, it appears unlikely that partial C2 deficiency is a disease‐susceptibility factor for the development of SLE. SLE has also been associated with acquired complement deficiency states, particularly in patients with reduced levels of C2 and C4 caused by the effects of inherited C1 inhibitor deficiency. The presence of the autoantibody, C3 nephritic factor, which causes C3 consumption, has also been associated with the presence of SLE. These conditions are discussed briefly below. C1 inhibitor deficiency may be hereditary (hereditary angioedema) or acquired. Hereditary C1 inhibitor deficiency is transmitted as an autosomal dominant trait and heterozygous individuals experience recurrent subcutaneous and submucosal swelling (recently reviewed in [44]). In normal circumstances, this inhibitor, a serine proteinase inhibitor (serpin), binds and inactivates enzymatically active C1r and C1s. Deficiency results in uncontrolled fluid phase classical pathway activation and consequently reduced levels of both C4 and C2. Following the observation that homozygous C4 and C2 deficiency predisposes to the development of SLE, it might be predicted that individuals with C1 inhibitor deficiency may have an increased likelihood of developing lupus and this does appear to be the case [45–52]. The incidence of SLE in individuals with hereditary C1 inhibitor deficiency has been estimated at approximately 2% [46]. C3 nephritic factor is an IgG autoantibody directed against a neo‐epitope on the fluid phase and cell‐bound alternative pathway C3 convertase (C3iBb and C3bBb, respectively) [53]. The binding of C3 nephritic factor to the C3 convertase renders it resistant to inactivation by factor H. The consequent uncontrolled alternative pathway activation results in secondary C3 deficiency. C3 nephritic factor is most commonly associated with partial lipodystrophy and MPGN type II. However, associations between C3 nephritic factor and SLE have been reported [54–58]. In these reports the onset of SLE was between 2 and 24 yr after the onset of partial lipodystrophy or nephritis. The significance of this association is uncertain. It may be that common factors cause both the development of the C3 nephritic factor and SLE. However, it is rare to see C3 nephritic factor as part of the spectrum of SLE autoantibodies. Alternatively, prolonged C3 deficiency caused by the C3 nephritic factor, may predispose to the development of SLE. However, as discussed above, SLE is rare in homozygous C3 deficiency. Furthermore, homozygous factor H deficiency, which also results in severe C3 deficiency, has only been associated with SLE in one individual who was also found to have heterozygous C2 deficiency [59]. Similarly, homozygous factor I deficiency, again resulting in secondary C3 deficiency, is predominantly associated with recurrent pyogenic infections but not SLE [8]. The measurement of complement antigenic levels and functional activity in serum is commonly used as a marker of disease activity in SLE. During periods of active disease, serum complement activity is usually reduced. Typically, levels of the classical pathway components (C1q, C2, C4) are low and, especially in patients with severe disease, may be accompanied by a reduction in C3 levels. However, in cohorts of patients with SLE, levels of individual complement components only approximately correlate with disease activity and levels of C4 may remain low when the patient is well [60]. This may partially be explained by the fact that the level of any serum protein is related to the rate of synthesis as well as catabolism. Determinants of protein synthetic rate are not well understood and studies have shown that hypercatabolism of C3 in disease may be associated with increased or decreased synthetic rates [61–63]. This limits the value of the simple measurement of protein concentration in serum. The activation or breakdown products of complement proteins have been assayed in an attempt to circumvent this problem. Levels of a variety of such products have been reported to correlate with disease activity more strongly than simple measurements of total C3 and C4 levels. Examples of assays for complement activation products in SLE include C3a [64], iC3b [65], C3d [66] and C4d [67]. Furthermore, in pregnancy, where increased C3 synthesis occurs, a reduction in the ratio of total haemolytic activity (CH50) to the alternative pathway activation product Ba, has been shown to be the most reliable complement assay in distinguishing active lupus from pre‐eclampsia [68]. However, complement activation products are highly unstable and usually have a very short half‐life in plasma. At present, all complement assays are at best a very crude surrogate marker of disease activity in SLE. It should also be appreciated that other factors, independent of disease activity, may affect complement levels in lupus patients. The most important are a series of autoantibodies to complement proteins which include: anti‐C1q autoantibodies [69], anti‐C1 inhibitor antibodies [70, 71], classical pathway nephritic factor (an autoantibody to the classical pathway C3 convertase, C4b2a) [72] and C3 nephritic factor [53]. It has been suggested that the measurement of complement activity may identify patients at risk of developing lupus nephritis. An early study demonstrated that normal C3 levels were rarely detected in the presence of active nephritis [60]. More recently, the presence of anti‐C1q antibodies in SLE has been found to correlate strongly with the presence of proliferative nephritis with a positive predictive value of 50–58% and a negative predictive value of 96.5–100% antibodies are IgG autoantibodies that are directed against on the region of the C1q These found in of lupus patients a significant with levels of C1q, C3 and C4 in anti‐C1q have been shown to in SLE and, in contrast to in appear to increase to Furthermore, these antibodies have been from of lupus patients with proliferative nephritis in than that of serum the hypothesis that play a direct role in the pathogenesis of the nephritis. There is one extremely important clinical association of hypocomplementaemia in SLE. with hypocomplementaemia are at particular risk of developing with such as and These patients can be to be because the in to and also results in reduced clearance of these There is a strong case that such patients, to and be for both and of the important physiological of the classical pathway of complement activation is the clearance of immune The of this in the pathogenesis of SLE and the role of complement in immune are discussed. 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