SLE and Serum Complement: Causative, Concomitant or Coincidental?

2.1. The Complement System and SLE

The complement system is a complex pathway in the immune system composed of proteins and receptors that improve the ability of antibodies and phagocytes to remove microbes and damaged cells from an organism. In addition to its function in combating infectious diseases, it also plays a key role in the inflammatory response prompted by immune complex deposition in numerous autoimmune diseases, including SLE.

Most complement proteins circulate in an inactive form and can be activated by three pathways: the classical pathway, the alternative pathway and the lectin pathway Fig. (2). Regardless of the pathway, cleavage of C3 and C5 ultimately occurs, producing C3a and C5a anaphylatoxins, C3b opsonins, and C5b for the creation of the membrane attack complex (MAC), which comprises fragments of C3b, iC3b and C3d that act as a bridge to phagocytic cells. There is also the production of chemotactic factors, inflammatory modulators [7-9] and antibody production, whereby antigens bind to C3d, which interacts with complement receptors and B cell receptors on B-type lymphocytes that activate, proliferate and produce antibodies [10]. The complement pathway then assists in clearance of immune complexes and cell lysis via the membrane attack complex and removal of apoptotic cells and debris via C1q, C4b and C3b, which enhance the ingestion of dead cells by phagocytes [11-13].

Numerous studies have demonstrated a decrease in complement proteins in patients with active SLE, for which quantifying complements have become a standard of care since the 1950s [14-16]. Inherited deficiencies of the complement system, however, demonstrate an independent predisposition of affected individuals to bacterial infections and SLE [17-19]. Complement deficiencies are said to comprise between 1 and 10% of all primary immune deficiencies [18], with an increased association of early complement pathway deficiencies (C1q, C2, C4) in autoimmune diseases [20, 21] like SLE and deficiencies in late complement pathway components (C5-C9) associated with infections, particularly pneumococcal, influenza, and Neisseria infections [22].

It is the loss of self-tolerance that results in autoantibody production in SLE patients with impaired clearance of apoptotic debris. C1q and other proteins typically assist in the removal of apoptotic material, which may explain why C1q deficiency has specifically been associated with impaired immune complex and apoptotic cell clearance [23]. Anti-C1q antibodies also bear a direct association with renal involvement in SLE and potential predilection for renal flares. The central pathway for complement activation in SLE is the classical pathway when activated by immune complexes, which present decreased serum levels of C1q (90-93%), C1r/s (50-57%), C4 (75%), and C2 (10%) (14-17). A more recent review of C1q-deficient patients identified the SLE phenotype to include discoid rash, oral ulcers and anti-smith antibodies with an unusually negative anti-dsDNA and less frequent arthritis [24, 25], implicating potential utility in screening dsDNA-negative cutaneous lupus with serum C1q levels.

While C3 may also be low in SLE, its levels tend to remain normal due to the inhibition of classical pathway activation by C4 binding protein [26]. Low levels of C3 are usually associated with low levels of Factor B, indicating an increased C3 turnover through involvement of the alternative pathway [27, 28].

C4 protein is encoded by two genes (C4A and C4B), each with different functions and different genes on chromosome 6 [29]. Both components demonstrate differences in binding reactivity, with C4B binding twice as effectively as C4A to antibody-coated RBCs but C4A more avidly binding to the protein-antigen complexes. Complete or homozygous deficiency of C4 [missing both C4A genes, reflecting homozygosity for human leukocyte antigen [HLA-B8 (DR3)] is uncommon in the general population but is one of the strongest genetic risk factors for SLE and may result in lupus-like disease in up to 80% of known affected individuals [30-32]. Of note, the most consistent HLA associations with SLE include HLA-DRB1*03:01 (DR3) and HLA-DRB1*15:01 (DR2) and their respective extended haplotypes in European populations [33]. Though rarely done in SLE, genetic testing is required to determine C4 genetic deficiency; levels of C4 may be 0, 25, 50, or 75 percent of normal, depending upon whether the specific individual inherits four, three, two, or one non-expressed allele(s), respectively. In these individuals, the C4 level is at borderline being low throughout life and, if SLE develops, the level of C4 tends to decline more than the baseline levels. Furthermore, because these patients will continue to show low or low-normal total C4, even with effective treatment of disease, our tendency to rely on declining C4 as a surrogate for SLE flares becomes limited. There are two explanations for this picture:

1. The disease is in remission and patient is C4A-deficient (i.e. lacks expressed C4A alleles) such that the patient will indefinitely have a low serum C4.

2. There is ongoing classical pathway activation due to active disease (it is important in this scenario to repeat C4 to ensure that the levels are, in fact, low and not due to lab error). As noted, the interpretation of a low serum C4 in a patient with SLE can be complex because reduced levels may be due to the complement consumption or deficiency of one or more alleles or even both scenarios in one patient. In addition, while inherited deficiencies of the complement system are characterized by the static absence of a single complement component, in consumption (as in SLE), there is a fluctuation over time and a decrease in multiple components of complement i.e. low C2, C3, and C4. In the face of uncertainty, a blood sample may be sent to a commercial laboratory specializing in complement tests. To determine the number of C4 alleles at the DNA level, the sample would need to be sent to a research laboratory where HLA typing can determine if the patient carries the HLA-B8, DR3 haplotype. Definitive reviews have been published on determining the number of C4 genes in a large number of normal controls and SLE patients [34-36].

The total hemolytic complement (THC or CH50) assesses the ability of serum to lyse sheep red blood cells sensitized with rabbit immunoglobulin M antibody. All components of the classical pathway are required to give a normal CH50, which makes it a useful tool for detecting a deficiency of the classical pathway. It does not assess the alternative pathway because factors B, D, and properdin are not required for the classical pathway. A very low or zero CH50 can result from a genetic deficiency of one or more complement proteins. Moderate reductions in CH50 are often seen in pathologic processes secondary to immune complex formation [37].

While complement activation is a hallmark of the pathogenesis of SLE with decreases associated with hematologic and renal disease [38], decreased complement levels are not consistently associated with flares. In fact, drawbacks of measuring complement levels to assess disease activity include:

1. Variations in complement protein levels due to the individual genetic polymorphisms.

2. Production and breakdown of complement proteins vary with SLE disease activity and individual synthesis response variability. C3 and C4 are also acute phase reactants, implicating an increased synthesis rate in response to inflammatory stimuli. Similarly, this increased synthesis rate may compensate for the increased breakdown demonstrated in SLE due to complement activation [39].

3. Serum complement levels may vary when compared to the tissue levels as demonstrated previously with glomerular basement membrane deposition of complement [40, 41].

4. Autoantibodies to C1q may activate complement in vivo and activate the complement system regardless of disease activity.

Additional studies measuring complement activation products allow for a different, albeit controversial, approach to monitoring SLE disease activity. Elevated levels suggest that SLE (or other diseases in question) is active, while low levels imply that consumption is greater than synthesis [42]. While this is older literature, a functional assay was suggested at the time for all patients with SLE. Unfortunately, direct measurement of complement activation products is not routinely available, is short-lived and may also be affected by activation during the procurement and freeze thawing. Recent data, however, has identified serum cell-bound complement activation products (CB-CAPS) with half-lives as long as the hematopoietic cells with which C3d and C4d (erythrocytes EC4d, B-type lymphocytes BC4d) are bound [43]. These CB-CAPS are identified in most patients with SLE and have demonstrated greater sensitivity and specificity compared to the use of low serum complements for the diagnosis of SLE [6, 44]. Similar studies have demonstrated that elevated levels of complement split products, particularly those of the alternative and terminal pathway activation, may more accurately reflect disease activity than conventional monitoring of complement C3 and C4 in predicting an impending SLE flare [41, 45]. More studies are indicated to clarify this concept and are ongoing with CB-CAPS demonstrated to be quite specific (80-90%) for and present even in mild SLE [43, 44].

The Avise Lupus test was developed in response to the limitations of current diagnostic tests for SLE [6]. It incorporates objective measurement of CB-CAPs and autoantibodies associated with connective tissue diseases. Validation studies have demonstrated that the test has high sensitivity and specificity, and even higher sensitivity than the American College of Rheumatology (ACR) score [6]. This supports earlier findings that CB-CAPs are valuable biomarkers of SLE and that the Avise Lupus test can be an effective supplement in the diagnosis of SLE when clinical and immunological features of SLE are insufficient [46, 47].