Systemic lupus erythematosus is arguably the most clinically and serologically diverse autoimmune disease. This article highlights the biomarkers helpful in diagnosing this disease. The authors’ own research is presented.
Systemic lupus erythematosus (SLE) is arguably the most clinically and serologically diverse autoimmune disease. Currently available information suggests that intricate interactions between environmental factors, hormonal factors, and disease susceptibility genes may predispose an individual to develop aberrant immune responses leading to SLE. Such aberrant responses, characterized by polyclonal activation of autoreactive lymphocytes, autoantibody production, immune complex formation, and complement activation, lead to acute and chronic inflammation in various tissue and organ systems. Owing to its complex etiopathogenesis, heterogeneous presentation, and unpredictable course, SLE remains one of the greatest challenges to both investigators and physicians. Currently, the diagnosis of SLE primarily is based on the presence or absence of American College of Rheumatology (ACR) criteria. Disease activity in SLE patients often is assessed using indices such as the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), the Systemic Lupus Activity Measurement (SLAM), and the British Isles Lupus Assessment Group (BILAG) index. The lack of easy-to-measure, reliable, and specific biomarkers for SLE not only hampers precise assessment of disease activity and accurate evaluation of response to treatment, but also impedes the development of novel therapeutics targeting key pathogenic factors. Therefore, there is an urgent need for reliable, specific biomarkers in not only lupus patient care, but also in research.
Complement and SLE: the historical bond
The complement system has been linked more intimately to SLE than to any other human disease. The involvement of complement proteins in the etiopathogenesis of SLE has been recognized and investigated for decades. In the current era of biomarkers and targeted therapeutic approaches, investigators’ interest in the complement system has been reinvigorated.
The Complement System
The complement system comprises a group of plasma and membrane-bound proteins that form three distinct—classical, alternative, and lectin—pathways designed to protect the host against invasion of foreign pathogens. The classical pathway is activated by antibodies or antigen–antibody complexes (immune complexes). Distinct from the antibody-dependent classical pathway, the alternative pathway is initiated when spontaneously generated complement components bind to surfaces of invading organisms or self-tissues, whereas the lectin pathway is triggered when mannose-binding lectins attach to polysaccharides uniquely expressed on the surface of microorganisms. Overall, the cascading reaction of complement activation generates proteolytic fragments that are capable of attracting inflammatory cells, inducing production and release of inflammatory mediators, and tagging invading organisms to be promptly phagocytosed by neutrophils and monocytes. Consequently, activation of the complement system may lead to not only physiologic immune responses but also to pathologic immune–inflammatory tissue damage. An unfortunate example of the latter is the myriad disease manifestations in SLE.
C3 is the most abundant protein in the complement system and also the indispensable molecule involved in all three activation pathways of the complement system. C4 is the second most abundant component and is essential for the classical and lectin pathways. C3 and C4 are synthesized predominantly in the liver and undergo post-translational modification to become 3-chain proteins linked by disulfide bonds. Both C3 and C4 contain an internal thioester site that is located within the C3d and C4d region in the α chain of each respective parental molecule. During activation of the classical pathway, proteolytic cleavage of C4 by C1s generates a small peptide C4a and a major fragment C4b. C4b contains the activated, highly reactive thioester that can readily interact with hydroxyl or amino groups on the receptive surface (eg, pathogens, host cells, or immune complexes) to form covalent ester or amide linkages. Target-bound C4b then cleaves C2, resulting in the formation of C4bC2a complexes that function as the C3 convertase. Like C4, C3 is proteolytically cleaved during activation of the complement system, yielding a small peptide C3a and a large fragment C3b. Similar to C4b, C3b is capable of binding covalently to acceptor molecules on target surfaces via ester or amide linkages. C4b and C3b may be cleaved further by Factor I, yielding ultimately C4d and C3d.
Once C3b binds covalently to target cells, it recruits C5 and cleaves the latter into C5a and C5b. Binding of C5b to target cells initiates the formation of the C5b-9 membrane attack complexes (MAC). Perhaps in a dose-dependent manner, MAC inserted into the membrane can either activate (at sublytic levels) or cause lysis of target cells. In contrast to their larger, enzymatically active counterparts, the smaller fragments C4a, C3a, and C5a (collectively referred to as anaphylatoxins) are potent chemotactic factors that are capable of recruiting leukocytes into inflamed tissues. Moreover, by binding to specific receptors expressed on most infiltrating leukocytes and endothelial cells, these anaphylatoxins can cause activation and release of numerous inflammatory mediators from host cells, thereby aggravating and perpetuating inflammatory tissue damage.
Soluble Complement Proteins as Lupus Biomarkers
Because antibody/immune complex-triggered activation of the complement system is thought to play an important role in the pathogenesis of SLE, one might expect complement proteins to be consumed to an extent proportional to the disease activity. Thus, measures of complement C3 and C4 have historically been viewed as gold standard laboratory tests for SLE. Many physicians consider decreases in serum C3 and C4 levels as indications of increased inflammation and SLE disease activity. There are several drawbacks to this approach, however. First, there is a wide range of variation in serum C3 and C4 levels among healthy individuals, and this range overlaps with the range observed in SLE patients. Second, standard laboratory tests measure the concentration of parental C3 and C4 molecules rather than products of activation. Third, acute-phase response during inflammation may lead to an increase in C4 and C3 synthesis, which can balance the activation and increased consumption of these proteins. Fourth, partial deficiencies of C4, which are commonly present in both the general population and SLE patients, may result in lower than normal serum C4 levels because of decreased synthesis rather than increased complement activation or active SLE. As a result of these confounding factors, there have been conflicting conclusions regarding the value of serial measurement of serum C4 in monitoring disease activity in SLE patients. Some studies have found serum C4 and C3 levels valuable in this regard, while others have found C4 and C3 levels to remain normal during SLE flares. These conflicting results suggest that current standard tests, based on serum levels of the native form of complement proteins, are inadequate to accurately and promptly detect SLE disease flares. During the past several years, other investigators have explored the potential for measurement of soluble complement activation products such as C3a, C5a, and C4d to serve as biomarkers in SLE. Despite some intriguing observations, serum levels of complement activation products have not replaced measurement of native C3 and C4 as gold standards.
Cell-bound complement biomarkers: complement measures and lupus revisited
Given the less-than-satisfactory performance of soluble complement components as lupus biomarkers, there is strong incentive for developing alternative complement-based biomarkers.
Rationale for Cell-Bound Complement Biomarkers
Complement proteins are abundant in the circulation and in tissues. Besides floating freely as soluble proteins, both the parental molecules and their activation derivatives can readily interact with cells circulating in the blood (eg, erythrocytes and lymphocytes) or tissues (eg, endothelial cells). Conceivably, complement activation products generated during SLE flares may attach to various circulating and tissue cells and alter physiologic functions of those cells. The rationale for exploring cell-bound complement biomarkers is as follows. First, most soluble complement activation products are easily subjected to hydrolysis in circulation or in tissue fluids and thus are short-lived. Second, activation products derived from C3 and C4 contain thioester bonds capable of covalently attaching to circulating cells and may decorate the surfaces for the lifespan of those cells. Third, many hematopoietic cells express receptors for proteolytic fragments generated upon complement activation. Fourth, products of C4 activation are known to be present on surfaces of erythrocytes of healthy individuals. Therefore, cell-bound complement components have the potential to be long-lived and may perform more reliably than soluble complement proteins as biomarkers for SLE.
Experimental Studies of Cell-Bound Complement Activation Products
Recent studies in the authors’ laboratory have been focused on discovery and validation of cell-bound complement activation products (CB-CAP) as potential lupus biomarkers. Using flow cytometry assays, a unique CB-CAP phenotype of circulating blood cells that is highly specific for SLE has been identified ( Fig. 1 ).
Considering the physiologic abundance and localization of erythrocytes, the authors have hypothesized that erythrocytes, circulating throughout the body and hence having easy assess to products derived from systemic as well as local activation of the complement system, may serve as biologic beacons of the inflammatory condition in vivo (and hence the disease activity) in patients with SLE or other inflammatory diseases. To verify this hypothesis, the first CB-CAP study was a cross-sectional investigation examining erythrocyte-bound C4d (E-C4d) levels in patients with SLE (n = 100), patients with other inflammatory and immune-mediated diseases (n = 133), and healthy controls (n = 84). In light of the previous reported association of low erythrocyte-complement receptor 1 (E-CR1) levels in SLE, E-CR1 was determined simultaneously. This study demonstrated unambiguously for the first time that patients with SLE have significantly higher levels of E-C4d (specific mean fluorescence intensity [SMFI] = 24.6 plus or minus 28.5) as compared with patients with other diseases (SMFI = 9.3 plus or minus 6.5; P <.001) and healthy individuals (SMFI = 6.7 plus or minus 5.3; P <.001).
A subsequent study took advantage of the knowledge that erythrocytes develop from hematopoietic stem cells in the bone marrow and emerge as reticulocytes, which then maintain distinct phenotypic features for 1 to 2 days before fully maturing into erythrocytes. Reticulocytes, if released into the peripheral circulation during an active disease state, may immediately be exposed to and bind C4-derived fragments generated from activation of the complement system. Therefore, it was hypothesized that the levels of C4d bound on reticulocytes (R-C4d) may effectively and precisely reflect the current disease activity in a given SLE patient at a specific point in time. The results of a cross-sectional study involving 156 patients with SLE, 140 patients with other autoimmune and inflammatory diseases, and 159 healthy controls showed that:
R-C4d levels of patients with SLE (SM[median]FI = 5.5 plus or minus 9.0; range: 0.0 to 66.8) were significantly higher than those of patients with other diseases (SMFI = 1.8 plus or minus 2.0; range: 0.0 to 17.6) or healthy controls (SMFI = 1.4 plus or minus 0.7; range; 0.0 to 4.7)
R-C4d levels fluctuated over time in patients with SLE and correlated with clinical disease activity as measured by the SLEDAI and SLAM indices.
Additional studies explored the possibility that CAP also may bind to nonerythroid lineages of circulating cells such as platelets and lymphocytes. A cross-sectional comparison of platelet-bound C4d (P-C4d) in patients with SLE (n = 105), patients with other inflammatory and immune-mediated diseases (n = 115), and healthy controls (n = 100) showed that abnormal levels of C4d were present on platelets in 18% of SLE patients, 1.7% of patients with other diseases, and 0% of healthy controls. More recently, the authors tested their hypothesis by flow cytometric analysis of C4d on T and B lymphocytes (referred to as T-C4d and B-C4d, respectively) from patients with SLE (n = 224), patients with other diseases (n = 179), and healthy controls (n = 114). Remarkably, both T-C4d and B-C4d levels were significantly and specifically elevated in SLE patients (SM[median]FI = 12.1 plus or minus 20.5 [T-C4d] and 49.0 plus or minus 73.2 [B-C4d]), as compared with healthy controls (SMFI = 1.7 plus or minus 1.0 [T-C4d] and 8.8 plus or minus 8.5 [B-C4d]; both P <.001) and patients with other diseases (SMFI = 2.5 plus or minus 3.0 [T-C4d] and 14.7 plus or minus 26.8 [B-C4d]; both P <.001).
Collectively, these studies strongly suggest a CB-CAP phenotype that is highly specific for patients with SLE. Moreover, we have noticed that high levels of C4d are not necessarily concurrently present on erythrocytes, reticulocyte, platelets, and lymphocytes of a given SLE patient at a particular time (Liu and colleagues, unpublished data). These findings suggest that binding of CAP to circulating blood cells does not merely reflect complement activation occurring during SLE disease flares, but may also reflect specific cellular and molecular mechanisms in lupus pathogenesis.
Cell-bound complement biomarkers: complement measures and lupus revisited
Given the less-than-satisfactory performance of soluble complement components as lupus biomarkers, there is strong incentive for developing alternative complement-based biomarkers.
Rationale for Cell-Bound Complement Biomarkers
Complement proteins are abundant in the circulation and in tissues. Besides floating freely as soluble proteins, both the parental molecules and their activation derivatives can readily interact with cells circulating in the blood (eg, erythrocytes and lymphocytes) or tissues (eg, endothelial cells). Conceivably, complement activation products generated during SLE flares may attach to various circulating and tissue cells and alter physiologic functions of those cells. The rationale for exploring cell-bound complement biomarkers is as follows. First, most soluble complement activation products are easily subjected to hydrolysis in circulation or in tissue fluids and thus are short-lived. Second, activation products derived from C3 and C4 contain thioester bonds capable of covalently attaching to circulating cells and may decorate the surfaces for the lifespan of those cells. Third, many hematopoietic cells express receptors for proteolytic fragments generated upon complement activation. Fourth, products of C4 activation are known to be present on surfaces of erythrocytes of healthy individuals. Therefore, cell-bound complement components have the potential to be long-lived and may perform more reliably than soluble complement proteins as biomarkers for SLE.
Experimental Studies of Cell-Bound Complement Activation Products
Recent studies in the authors’ laboratory have been focused on discovery and validation of cell-bound complement activation products (CB-CAP) as potential lupus biomarkers. Using flow cytometry assays, a unique CB-CAP phenotype of circulating blood cells that is highly specific for SLE has been identified ( Fig. 1 ).
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