Making Sense of Inflammation

The Complement System

The complement system was identified over 120 years ago and has been the study of intense investigation in term of its biochemical mechanisms of action, regulatory mechanisms and role in 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 found in blood and on cell membranes. Complement exists in a quiescent state until it is activated. Activation of complement consists of initiation of a proteolytic cascade. Many complement proteins exist as inactive pro-proteins and once complement activation occurs a series of sequential proteolytic events occur that release biologically active molecules that promote phagocytosis and lysis of microbes and provoke an inflammatory reaction involving recruitment of immune cells, increased vascular permeability, vasodilatation, etc.

There are three known pathways that initiate complement activation: 1) classical pathway; 2) alternative pathway and 3) the lectin pathway (Figure 1).  Each pathway is unique in the molecules that trigger activation and the mechanism and proteins involved in regulation.  Regardless of the initiation event, all three pathways converge on complement (C3). Proteolytic cleavage of C3 is necessary for generating all of the effector mediators of complement function (Figure 1).  The two proteolytic cleavage products of C3 activation are C3a, an anaphylotoxin, and C3b, an opsonin. C3b is further processed into other active and inactive products such as C3a, iC3b, etc (Figure 2).

Figure 1

Figure 1

Figure 2

Figure 2

Importance of complement in human health and disease

The complement system is a major component of the innate immune system and a major effector system of the acquired immune system.  The complement system is composed of approximately 40 proteins, each of which is involved in a highly regulated cascade of biochemical processes in response to pathogens, cancer and injury. Activation of the complement cascade is essential for the body to recover from infectious diseases, eliminate cancer cells, and remodel tissue following damage. Alternatively, complement is well-known to play a prominent role in the pathogenesis of many autoimmune and inflammatory diseases and dysregulation of complement can cause or worsen damage to end-organs such as kidneys, the nervous system, joints, etc. 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 some laboratories also perform functional assays such as complement-mediated hemolytic assays (e.g. CH50 or AP50) to measure levels of complement activity in the blood. The purpose of these measurements are usually related to determination of suspected complement deficiency (Mollnes et al. Mol Imm 2007).

Complement testing has more recently been performed to assess the inflammatory state in patients with autoimmune inflammatory diseases (e.g. SLE, vasculitis, MPGN, PNH, aHUS, HAE, etc.) because it has become clear that complement dysregulation is a major pathophysiological mediator of the inflammation.

Clinical utility of complement testing: (Why do clinicians want to know about complement activation in their patients?)

Patients with known or suspected autoimmune diseases can present with various degrees of inflammation. The spectrum can range from totally quiescent 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. Biochemical tests which measure ‘inflammatory biomarkers’ such as erythrocyte sedimentation rate (ESR) or c-reactive protein (CRP) levels are often used, but are very non-specific and can be elevated in conditions unrelated to complement-mediated inflammation. As one measure of the clinical importance of complement, a reported 90% of rheumatologists order C3 testing for their SLE patients (Donald et al. Arthritis and Rheum 1998).

Measurement of complement activation

Complement activation can occur through three distinct pathways, each of which converges on the central protein of the complement system, C3. Most of the complement-related pathology requires C3 activation. Therefore, determining the presence and activation status of C3 in biological specimens can be used as a reflection of a particular patient’s inflammatory status, especially if evaluated in conjunction with other diagnostic tests. Thus physicians who manage patients in whom complement-mediated inflammation may be occurring want to know the extent of complement activation and how it’s changing 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 (e.g., disease, infection, or tissue injury), the native protein is processed into various proteolytic cleavage products (‘split products’) including C3a, iC3b,C3c, C3d, etc. 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. Simplistically, the idea is that as complement activation occurs C3 is consumed and C3 levels drop, which serves 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 detect the native C3 protein as well as some many of the C3 split products. Levels of native C3 in the blood are a function of three factors: 1) rate of synthesis by the liver, 2) rate of non-specific catabolism (half-life), and 3) consumption via complement activation whereby C3 is converted into specific C3 split products. 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, liver function, etc. Second, C3 is a known acute phase reactant, i.e. the liver increases synthesis of C3 non-specifically in response to bodily insults such as infection or injury. Imagine a patient with ongoing inflammation. C3 synthesis increases in response the to the inflammatory stimulus.  In parallel complement activation occurs which causes consumption of C3. The end result could be no net change in C3 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 the most abundant protein in human blood with levels that can exceed 1000 micrograms per milliliter of blood. Significant and physiologically relevant activation of the complement system may involve consumption of a few micrograms of C3 and production of C3 split products in micrograms/ml. These levels of C3 split products are 1000-fold lower than levels of total C3 and thus small or even relatively insignificant changes in C3 levels could occur while C3 split products are increasing by 10-fold or greater. In summary, although measurement of C3 levels in patients is currently a routine clinical laboratory practice, measurement of C3 levels can lead to both false positive (low C3 levels are due to poor low  synthesis rather than complement activation) and false negative  (normal C3 levels occur in the presence of ongoing complement activation) results.

Measurement  of complement split products.
Why measurement of complement split products is a better assessment of complement activation and ongoing inflammation.

As mentioned above, when C3 is activated the native protein is processed into various split products including C3a, iC3b,C3c, C3d, etc.. Other downstream products of complement activation such as C4d and C5a also increase. C3 split products are not synthesized directly; and they can only be generated by specific activation of C3 and are not generated by non-specific degradation. Therefore the presence of C3 split products is direct evidence of complement activation. Many studies have shown that plasma concentrations of complement split products are increased before or during clinical exacerbation of SLE and levels often correlate with disease activity (Buyon et al. Arthritis and Rheum, 1992; and others). Other studies have not shown such a correlation. However, these types of studies are plagued by the problem of unreliable results due to poor specimen handling and possible auto activation.

What are the critical issues in measurement of C3 split products?

The utility of measuring C3 split products as a measure of complement activation is affected by several factors. First, the half-life of the split product is important. A long half life (e.g. many hours) would reduce the ability of the level of the split product to be used to assess ongoing inflammation because high levels could be indicative of past complement activation. Too short a half life (e.g. several minutes) would reduce the sensitivity of the split product because it would take high levels of complement activation to result in a significant rise in levels of a split product with a short half life (fast catabolic rate). The optimal half life would be >30 minutes-<2 hours. Second, tests for a split product must be highly specific for that split product. Because all C3 split products are derived from the same protein they share common antigenic epitopes. For immunoassays, epitopes unique to the split product need to be available to make antibody specific for the split product.

Advantages of iC3b (vs. other split products)
Why iC3b is an good ideal analyte for assessing complement activation.

The C3 split product  iC3b satisfies the major criteria for a good biomarker of complement activation. 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 that are unique to iC3b and not found on native C3 or on other C3 split products. This has enabled the isolation of monoclonal antibodies that are specific to iC3b (NEO-iC3b) and the development immunoassays that can detect iC3b with a high degree of specificity (Tamerius, J.D., et al. J. Immunol. 135, 2015–2019). As mentioned above, iC3b is not synthesized directly and 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 very specifically and is a very direct measure of ongoing complement activation.

Specificity of reagents (for iC3b)

Using highly specific Mabs monoclonal Abs to iC3b neoantigens, the COMP ACT™ test measures iC3b with high specificity and minimal cross-reactivity (Tamerius et al. J. Immunol. 1985). There is a high correlation between the COMP ACT test (Kypha LFA) and commercial ELISA for iC3b (under conditions in which autoactivation 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 SLE and other inflammatory diseases (Manderson AP, et al. Annu Rev Immunol. 2004;22:431-56). There are several [CS1] studies that directly demonstrate the value of iC3b as a clinical biomarker for complement-mediated inflammation. Prior to the development of the iC3b LFA test it was difficult to measure iC3b due to problems associated with autoactivation during specimen handling and shipment and thus it was very difficult to obtain reliable results. Negoro et al. measured levels of iC3b using a monoclonal antibody specific for iC3b-NEO in an ELISA assay to measure complement activation in SLE patients (Negoro et al. Arthritis & Rheumatism 32, 1233, 1989) The plasma iC3b-NEO level in 40 untreated patients with active SLE was significantly higher than that in 36 normal subjects. The plasma iC3b-NEO level was highly correlated with clinical disease activity and it was the parameter most closely correlated with renal histologic activity in lupus nephritis.

Expert Opinions

Comments by Rheumatologist Dr. Vibekea Strand:

“Although C3 and C4 levels are routinely used to monitor complement activation, they may not accurately reflect such activation and lack sensitivity to identify lower levels of activation and/or when consumption exceeds production due to changes in underlying production of complement components in the liver. The normal ranges for both functional assessments and immunoassays are wide and require a substantial change before it can be ascertained that complement consumption exceeds production.

Furthermore C3 is an acute phase reactant, thus production in the liver is increased by inflammation, and decreased when androgens or IL-6 inhibitors are administered. IC3b is a good biomarker of complement activation, better than C3a or C3b, because it is not cleared as rapidly. With a longer half life of approximately 90 minutes, it is preferable to C3c or C3d with half-lives of 24 and 50 hours, respectively, and therefore represents a better assay of acute complement activation/consumption. Coupling accurate point of care assays for both C3 and iC3b simultaneously can therefore distinguish between increases or decreases in C3 production in the liver due to inflammation and/or androgen or IL-6 inhibitor administration and increased activation /consumption of complement due to inflammatory conditions such as SLE or vasculitis. Furthermore, the accuracy of a point of care test will alleviate issues regarding in vitro complement activation associated with sample handling and storage as well as assay methods. It has been previously demonstrated that EDTA is not able to prevent complement activation over time in stored samples.”

Comments by Complement Experts:

Tom Mollnes, MD, PhD, Patsy Giclas, PhD, Haifeng Wu, MD – Clinical Pathology Labs
*All have intimate knowledge of the value and challenges of measuring C3 split products

George Tsokos, MD – rheumatology and trauma/critical care
*Could speak to broad clinical utility in support of underlying mechanism of pathophysiology

John Atkinson, MD – rheumatology
*Global KOL in complement

Spero Cataland, MD – hematology
*aHUS and TTP perspective, firm understanding of value and challenges of measure C3 split products

Michael Holers, MD – rheumatology
*Complement KOL very savvy in business and commercialization

E. Sander Connolly, MD – neurosurgery

 

References

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

http://labtestsonline.org/understanding/analytes/complement-levels/tab/test/

http://www.nationaljewish.org/professionals/clinical-services/diagnostics/adx/about-us/lab-expertise/complement/

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.
[CS1]Would it be “several” if we also included studies that measured C3b/c?
[CS2]Good place to reiterate complement’s broad underlying role in the “mechanism of pathophysiology”
[CS3]List them all