The Complement System
The complement system was identified over 120 years ago and has been the subject of intense investigation to understand its biochemical mechanisms of activation, host defense functions and role in the pathophysiology of human disease. The term ‘Complement’ is used to describe an arm of the innate immune system that serves to help the body eliminate microbial pathogens and remove cellular debris associated with tissue injury. Complement comprises over 40 proteins, receptors and regulatory molecules found in blood and on nearly all cell types in the body. Although complement requires activation to mediate its biological functions, the system is always primed to allow rapid activation. Immune complexes, bacteria, viruses and other pathogens can activate complement through one of several pathways, each of which initiates a proteolytic cascade. The complement proteins in these cascades are present as inactive pro-proteins and once cleaved generate the formation of multi-molecular enzyme complexes called “convertases” that cleave the two main proteins in the complement system C3 and C5 (Figure 1). Complement activation generates biologically active molecules that contribute to inflammation, phagocytosis and lysis of microbes.
There are three main complement activation pathways: 1) the classical, 2) alternative and 3) lectin pathways (Figure 1). Each pathway utilizes different proteins to generate the convertases, however, regardless of the activation event, all three pathways converge on the complement protein C3. Proteolytic cleavage of C3 is required for generating all of the effector functions of the complement system (Figure 1).
The two proteolytic cleavage products of C3 activation are C3a, an inflammatory molecule, and C3b, which attaches to pathogens and targets them for elimination. C3b is further processed into other activation fragments, including iC3b, a biomarker of complement activation employed by Kypha (Figure 2).
Importance of complement in human health and disease
The complement system is a major component of both the innate and adaptive immune systems. Although activation of complement is essential for elimination of pathogens and a number of other important functions, it is well known to play a prominent role in the pathogenesis of many autoimmune and inflammatory diseases, such as lupus, rheumatoid arthritis, stroke and acute and chronic infection. Furthermore, dysregulation of complement can cause or worsen damage to organs and tissues such as kidneys, the nervous system, and joints. In fact, much of the tissue damage seen in autoimmune and chronic inflammatory diseases is caused by excessive or unregulated activation of complement.
Laboratory measurement of Complement Activity
Clinical laboratories routinely measure levels of C3, C4 and C1-inhibitor by immunoassays such as ELISA, and perform functional assays such as hemolytic assays (e.g. CH50 or AP50) to measure levels of complement activity in blood. The purpose of these measurements is to determine and quantify suspected complement deficiency. Complement testing has more recently been performed to assess the inflammatory state in patients with autoimmune inflammatory diseases (e.g. lupus, vasculitis, paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome) because it has become clear that complement dysregulation is a major pathophysiological mediator of inflammation in these conditions.
Clinical utility of complement testing: Why do clinicians need to know about complement activation in their patients?
Patients with known or suspected autoimmune diseases can present with various degrees of inflammation. The spectrum ranges from mild to significant ongoing inflammation that can cause serious or even life-threatening organ damage. It is very difficult for physicians to determine the inflammatory state of a patient based on clinical signs and symptoms. Laboratory tests which measure ‘inflammatory biomarkers’ such as erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) levels are often used, but are often non-specific and do not measure inflammation in real time. As one measure of the clinical importance of blood complement levels, 90% of rheumatologists order C3 testing for their lupus patients.
Measurement of complement activation
Complement activation occurs through three distinct pathways, each of which converges on the central protein of the complement system, C3. Therefore, determining the presence and activation status of C3 in biological specimens can be used as a reflection of a patient’s inflammatory status, especially if evaluated in conjunction with other diagnostic tests. Physicians managing patients with complement-mediated inflammation want to know the extent of complement activation and how it changes over time.
A majority of the C3 protein in the human serum or plasma is in the native (or intact) state. However, when C3 is activated during infection, or as a result of tissue injury, the native protein is processed into various proteolytic cleavage products (‘split products’) including C3a, and iC3b. Since C3 is consumed during complement activation, measurement of C3 levels in blood (serum or plasma) is used by clinicians to assess the degree of complement activation occurring in an individual. The assumption is that C3 levels during complement activation are proportional to C3 consumption which is used as an indirect measure of the degree of ongoing inflammation.
Limitations of C3 levels as a measure of complement activation. Why measurement of C3 is a less than optimal way to measure complement activation.
Measurement of C3 levels in patients is currently a routine clinical laboratory practice. Nearly all C3 assays in use are based upon nephelometry and/or immunoturbidity platforms. These methods detect the native C3 protein as well as many of the C3 split products. Levels of native C3 in the blood are a function of three factors: 1) the rate of synthesis by the liver and other tissues, 2) the catabolic rate and, 3) consumption via complement activation. There are problems with using C3 levels for the purpose of assessing the inflammatory state of a patient. First, C3 levels can fluctuate according to an individual’s metabolic state and liver function. Second, C3 is a known acute phase reactant, i.e. the liver increases synthesis of C3 in response to infection or injury. Imagine a patient with ongoing inflammation. C3 synthesis increases in response to the inflammatory stimulus. In parallel complement activation occurs which causes consumption of C3. The end result could be no net change in C3 blood levels. Thus a normal C3 level in this patient would be a false negative result for a physician trying to use C3 levels to identify a patient with ongoing complement activation. An additional problem associated with using C3 levels to assess complement activation is related to the shear levels of C3. C3 is one of the most abundant proteins in human blood with normal levels ranging from 1.0 to 1.5 mg/ml. Thus even when significant and physiologically relevant C3 activation occurs, if the resulting change in C3 levels is small it may not be detected via measurement of total C3.
What are the critical issues in measurement of C3 split products? Why measurement of complement split products is a better assessment of complement activation and inflammation.
The presence of C3 split products is direct evidence of complement activation. In contrast to the native/intact C3 protein, C3 split products are present at much lower (e.g., 1-2 ug/ml) concentrations in the blood. C3 split products are a much more sensitive measure for detecting small changes in C3 activation. However, the utility of measuring C3 split products as a measure of complement activation and inflammation is affected by several factors, including sample integrity and protein stability, as well as the half-life of the split product. A long half-life (e.g. many hours) reduces the usefulness of the split product to assess ongoing inflammation because high levels could be indicative of past complement activation. Too short a half-life (e.g. several minutes) reduces the sensitivity of the split product as an inflammatory biomarker. The optimal half-life would be >30 minutes and <2 hours. In addition, tests for a split product must be highly specific since C3 split products are derived from the same protein and share common antigenic epitopes. For immunoassays, epitopes unique to the split product need to be available to make antibody specific for the split product.
Why iC3b is an ideal analyte for assessing complement activation.
The C3 split product iC3b satisfies the major criteria for a good biomarker of complement activation and inflammation. It has a half-life of 30-90 minutes and thus levels of iC3b reflect ongoing and recent complement activation. Secondly, iC3b contains well-characterized neo-antigens unique to iC3b and not found on native C3 or on other C3 split products. This enabled the isolation of monoclonal antibodies specific to iC3b (NEO-iC3b) and the development immunoassays that can detect iC3b with a high degree of specificity. As mentioned above, iC3b only occurs in the blood as a result of C3 activation. It is therefore a specific marker of complement activation. In summary, iC3b can be measured with high specificity and is a very direct measure of ongoing complement activation.
Specificity of reagents (for iC3b)
Using highly specific monoclonal Abs to iC3b neoantigens, the COMP ACT™ test measures iC3b with high specificity and minimal cross-reactivity. There is a high correlation between the COMP ACT test (Kypha LFA) and commercial ELISA for iC3b (under conditions in which auto-activation has been minimized by proper handling of the specimen).
Clinical validity of iC3b as a measure of complement activation
There is a large body of literature on complement activation and its importance in the pathophysiology of lupus and other inflammatory diseases. Furthermore, there are several studies that directly demonstrate the value of iC3b as a clinical biomarker for complement-mediated inflammation. Prior to the development of Kypha’s iC3b LFA test it was difficult to accurately measure iC3b due to problems associated with auto-activation during specimen handling and shipment. Many studies have shown that plasma concentrations of complement split products are increased before or during clinical exacerbation of several diseases, and levels often correlate with disease activity. It is well recognized that other studies have not shown such correlations; however, many of these types of studies are plagued by the problem of unreliable results due to poor specimen handling and processing protocols that result in spurious auto-activation.
Manderson AP, Botto M, Walport MJ. The role of complement in the development of systemic lupus erythematosus. Annu Rev Immunol. 2004;22:431-56. Review.
Fundamental Immunology, W.E. Paul (ed) Fifth Edition. 2003
Liu, CC and Ahearns, JM Complement and Tissue Injury in SLE in Basisi of Disease Pathogenesis: Systemic Lupus Erythematosus. Elsevier, 2011
Nilsson, B., Ekdahl, K.N., 2012. Complement diagnostics: concepts, indications, and practical guidelines. Clin. Dev. Immunol. 2012, 962702. doi:10.1155/2012/962702
Mollnes, T.E., Jokiranta, T.S., Truedsson, L., Nilsson, B., Rodriguez de Cordoba, S., Kirschfink, M., 2007. Complement analysis in the 21st century. Mol. Immunol. 44, 3838–3849. doi:10.1016/j.molimm.2007.06.150
Kirschfink, M., Mollnes, T.E., 2003. Modern Complement Analysis. Clinical and Vaccine Immunology 10, 982–989. doi:10.1128/CDLI.10.6.982-989.2003
Donald F, Ward MM. Evaluative laboratory testing practices of United States rheumatologists. Arthritis Rheum. 1998 Apr;41(4):725-9. PubMed PMID: 9550483.
Egner W. The use of laboratory tests in the diagnosis of SLE. J Clin Pathol. 2000 Jun;53(6):424-32. Review. PubMed PMID: 10911799; PubMed Central PMCID: PMC1731203.
Liu, C.-C., Manzi, S., Kao, A.H., Navratil, J.S., Ahearn, J.M., 2010. Cell-Bound Complement Biomarkers for Systemic Lupus Erythematosus: From Benchtop to Bedside. Rheumatic Disease Clinics of North America 36, 161–172. doi:10.1016/j.rdc.2009.12.003
Given, W.P., Edelson, H.S., Kaplan, H.B., Aisen, P., Weissmann, G., Abramson, S.B., 1984. Generation of C5-derived peptides and other immune reactants in the sera of patients with systemic lupus erythematosus. Arthritis Rheum. 27, 631–637.
Belmont, H.M., Hopkins, P., Edelson, H.S., Kaplan, H.B., Ludewig, R., Weissmann, G., Abramson, S., 1986. Complement activation during systemic lupus erythematosus. C3a and C5a anaphylatoxins circulate during exacerbations of disease. Arthritis Rheum. 29, 1085–1089.
Hopkins, P., Belmont, H.M., Buyon, J., Philips, M., Weissmann, G., Abramson, S.B., 1988. Increased levels of plasma anaphylatoxins in systemic lupus erythematosus predict flares of the disease and may elicit vascular injury in lupus cerebritis. Arthritis Rheum. 31, 632–641.
Kao AH, Navratil JS, Ruffing MJ, Liu CC, Hawkins D, McKinnon KM, Danchenko N, Ahearn JM, Manzi S. Erythrocyte C3d and C4d for monitoring disease activity in systemic lupus erythematosus. Arthritis Rheum. 2010 Mar;62(3):837-44.
Buyon, JP, Tamerius, J, Belmont, HM, Abramson, SB. Assessment of Diesease Activity and Impending Flare in Patients with Systemic Lupus Erythematosus Arthritis and Rheumatism 35:1028. 1992
Yang DH, Chang DM, Lai JH, Lin FH, Chen CH. Usefulness of erythrocyte-bound C4d as a biomarker to predict disease activity in patients with systemic lupus erythematosus. Rheumatology (Oxford). 2009 Sep;48(9):1083-7.
Navratil JS, Manzi S, Kao AH, Krishnaswami S, Liu CC, Ruffing MJ, Shaw PS, Nilson AC, Dryden ER, Johnson JJ, Ahearn JM. Platelet C4d is highly specific for systemic lupus erythematosus. Arthritis Rheum. 2006 Feb;54(2):670-4. Erratum in: Arthritis Rheum. 2006 Jun;54(6):2009. PubMed PMID: 16447243.
Manzi S, Navratil JS, Ruffing MJ, Liu CC, Danchenko N, Nilson SE, Krishnaswami S, King DE, Kao AH, Ahearn JM. Measurement of erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus. Arthritis Rheum. 2004Nov;50(11):3596-604.
Tamerius, J.D., Pangburn, M.K., Müller-Eberhard, H.J., 1985. Detection of a neoantigen on human C3bi and C3d by monoclonal antibody. “This neoantigen may therefore serve as a marker for C3b that covalently attached to cell membranes, immunecomplexes, or to othermacromolecules, and that subsequently was degraded to C3bi, C3dg. or C3d. “ J. Immunol. 135, 2015–2019.
Ueda, T., Rieu, P., Brayer, J., Arnaout, M.A., 1994. Identification of the complement iC3b binding site in the beta 2 integrin CR3 (CD11b/CD18). Proc. Natl. Acad. Sci. U.S.A. 91, 10680–10684.
Negoro, N., Okamura, M., Takeda, T., Koda, S., Amatsu, K., Inoue, T., Curd, J.G., Kanayama, Y., 1989. The clinical significance of iC3b neoantigen expression in plasma from patients with systemic lupus erythematosus. Arthritis & Rheumatism 32, 1233–1242.