The Complement System: A Key Player in Immunity
The complement system is an essential component of our innate immune defence and is crucial to the pathogenesis of many diseases. Activation of the complement system leads to a sequential cascade of enzymatic reactions resulting in the formation of the anaphylatoxins C3a and C5a that trigger an abundance of physiological responses that range from chemoattraction to apoptosis. Additionally, complement activation leads to the lysis of pathogens through formation of the Membrane Attack Complex (MAC; also known as C5b-9 or terminal complement component, TCC). MAC creates pores in cell membranes, this ultimately results in cell death.
Complement was initially recognized for its major contribution to innate immunity, where it facilitates a swift and potent response to invading pathogens. However, it is now increasingly evident that complement also plays an important role in the adaptive immune system. It is involved in the activation of T and B cells, which contribute to pathogen elimination and the maintenance of immunological memory, thereby preventing reinvasion of those pathogens. Not only is complement involved in innate and adaptive immunity, but it is also involved in tissue regeneration, tumor growth and diseases such as autoimmune and infectious diseases, kidney disease, neurodegenerative diseases and various types of cancer.
This text provides a general overview about the role and functions of the complement system: how it operates under healthy conditions, what happens when the system becomes dysregulated, and which diseases can arise from hyperactivation of the complement system. It also addresses how the medical and pharmaceutical fields are responding to these developments and highlights the importance of reliably measuring complement activation.
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Complement Pathway History
The complement system, now recognized as a vital component of innate immunity, has an intriguing history. The system was first described in the late 19th century by Jules Bordet. He observed that serum contained factors that enhanced the bacteriolytic activity of antibodies. Over time, this system was referred to by many names, including alexin, cytase, addiment, and opsonin of normal serum. Its activity was initially divided into a “midpiece” and an “endpiece.” A lot has changed since the foundational studies of the late 1800s and early 1900s, our understanding of complement has expanded. Today, we recognize multiple pathways – classical, lectin, and the alternative pathway – each contributing to a wide range of biological functions.
Complement Pathway Activation and Regulation
Complement is activated upon recognition of a pathogen or by damage-associated molecular patterns (DAMPs), such as apoptotic or necrotic cells. This activation initiates a cascade that helps mark and eliminate the threat. Complement activation is known to occur through three different pathways: classical, lectin and the alternative involving proteins that mostly exist as inactive zymogens that are then sequentially cleaved and activated. Each pathway is triggered by different molecular mechanisms.
The CP is initiated when C1q binds to antigen-associated IgM or IgG hexamers, forming immune complexes. The LP is activated upon recognition by specific molecules, including mannose-binding lectin (MBL), collectin-10, collectin-11, collectin-12, and ficolins-1, -2, or -3, which bind to microorganism-associated molecular patterns (MAMPs) or carbohydrate structures on damaged cells (Fig. 1). In contrast, the AP undergoes continuous low-level activation due to spontaneous C3 hydrolysis (Fig. 1), enabling rapid response on surfaces lacking appropriate complement regulation, typically non-host structures.
Irrespective of the initiating trigger all complement activation pathways converge at the proteolytic cleavage of C3, the most abundant complement protein found in blood, mediated by C3 convertases (Fig. 1). Upon activation, C3 undergoes conformational changes, resulting in the generation of fragments C3b and C3a. C3b is the larger fragment and functions as an opsonin by covalently attaching to amino or hydroxyl groups on target surfaces, such as microbial membranes. The smaller fragment, C3a, is an anaphylatoxin that exerts a range of immunomodulatory effects on both immune and non-immune cells. C3a plays a key role in amplifying complement activation. Through its interaction with the C3a receptor (C3aR), C3a promotes a range of biological effects, including phagocytosis, cell migration, chemotaxis, cytokine secretion, cell activation, and modulation of pattern recognition receptor (PRR)-driven responses. It thereby contributes to the amplification and full activation of the complement pathway.
When C3b is deposited at high density on a surface, it promotes the formation of C5 convertases. C5 activation generates C5a, an anaphylatoxin with strong pro-inflammatory and immunomodulatory effects, including the recruitment and activation of immune cells. Similarly to C3a which also acts as an anaphylatoxin. In addition to C5a, C5b is generated, which initiates the sequential assembly of the multiprotein, cell membrane-perforating complex C5b-C9 or MAC. Complement activation is rapidly initiated by environmental or microbial signals and is amplified primarily via the alternative pathway. While this enhances immune defence, excessive or uncontrolled activation can cause collateral tissue damage. To prevent this, the system is tightly regulated by soluble and membrane-bound proteins that serve as checkpoints to limit overactivation. For each type of activated fragment there is at least one inhibitor or inhibitory mechanism. These regulators act by accelerating the breakdown of C3 and C5 convertases or by preventing the formation of MAC. Most regulators are encoded by genes located in the RCA gene cluster. This cluster encodes for a family of structurally and functionally related proteins that negatively regulate the complement system to prevent uncontrolled activation and protect host tissues from damage.
For example, the classical pathway component C1 is regulated by C1-inhibitor (C1-INH) (Fig. 1), a plasma protein that stabilizes the inactive C1 complex and prevents spontaneous activation. More critically, C1-INH binds irreversibly to the active forms of C1r and C1s, thereby terminating their enzymatic activity. In addition, it also binds to MASPs, halting proteolytic activity in the lectin pathway.
A key regulator of the alternative pathway is factor H, the most prominent soluble regulator. Factor H is an important regulator, because C3 is continuously activated through the alternative pathway. It controls complement activation in the fluid phase by binding to host surfaces via interactions with glycosaminoglycans and oxidized self-ligands such as malondialdehyde-modified epitopes. It helps distinguish host tissue from foreign or damaged surfaces.
Regulation of C3b is mediated by Factor I, a serine protease that requires specific cofactors to exert its activity. These include Factor H, complement receptor 1 (CR1), and membrane cofactor protein (MCP/CD46). Similarly, C4b is inactivated by Factor I in the presence of its cofactor, C4-binding protein (C4BP) (Fig. 1). Through proteolytic cleavage, Factor I neutralizes C3b and C4b, preventing their interaction with downstream components of the complement cascade.
Membrane-bound regulators such as CD46, CD55 (decay-accelerating factor, DAF), and CD59 also play crucial roles. CD46 acts as a cofactor for factor-I-mediated cleavage of C3b and C4b. CD55 accelerates the decay of C3 and C5 convertases, while CD59 blocks the formation of MAC.
Despite these robust regulatory mechanisms, pathogens have evolved strategies to evade complement activity. These include hijacking host regulators like factor H, or producing viral mimics that interfere with complement activation.
Notably, properdin, an oligomeric plasma glycoprotein, is the only known positive regulator of the complement system. It stabilizes C3 and C5 convertases and initiates alternative pathway activation. It also has non-complement roles, such as interacting with natural killer cells.
Imbalance in complement activation and dysregulation
Complement activation is triggered by environmental or microbial signals and can be strongly and rapidly amplified via the alternative pathway. While this response is essential for host defence, insufficient regulation can lead to excessive activation, resulting in serious conditions such as thrombo-inflammation, immune dysregulation, vascular endothelial inflammation, and organ damage. To prevent overactivation, the complement system is tightly regulated. Inappropriate self-directed complement responses are prevented by the concerted action of fluid-phase and membrane-bound regulatory proteins that act as checkpoints to preclude prolonged or excessive complement activity as described above.
However, genetic or acquired deficiencies in complement inhibitors or their cofactors can disrupt the balance of complement regulation, potentially leading to uncontrolled activation, characterized by persistent opsonization, MAC-mediated cell lysis, and chronic inflammation. Such dysregulation is associated with a range of immune-mediated diseases affecting the eyes, kidneys, blood, and nervous system.
An example of imbalance due to a genetic deficiency is a mutation or polymorphism in the CFH-gen. This can lead to insufficient inhibition of spontaneous C3 activation. Resulting in excessive formation of C3 convertases and ultimately damage to self tissues. This genetic disorder is associated with diseases such as: Atypical hemolytic uremic syndrome (aHUS), Age-related macular degeneration (AMD) and C3 glomerulopathy. [2] More information about complement-driven disease mechanisms can be found in the FAQ section: What diseases are linked to complement system dysfunction?
Emerging Clinical Applications and Associated Challenges
Recent clinical milestones, such as the approval of eculizumab for generalized myasthenia gravis, underscore the system’s therapeutic versatility beyond traditional indications like atypical hemolytic uremic syndrome (aHUS). Complement modulation is now being explored in complex disorders ranging from neurodegeneration and autoimmunity to ischaemia-reperfusion injury, transplant rejection, and COVID-19-associated thromboinflammation.
One of the keys to innovation lies in the central inhibition of C3, which offers a broader blockade of the cascade compared to downstream C5 inhibition. C3-targeted approaches have demonstrated therapeutic potential in models of kidney transplantation, stroke, and severe lung inflammation. Importantly, localized complement modulation has proven effective while avoiding systemic immune suppression.
However, despite advances, several challenges remain. The complement system plays an important role in immune defence, but its activation is highly sensitive and can easily become dysregulated. In clinical settings, this sensitivity is both a strength and a challenge. Complement reacts quickly to infections, tissue injury, or immune dysregulation, making it a valuable tool for early diagnosis. Its rapid changes during disease progression or recovery also enable physicians to closely monitor treatment effects. Furthermore, because complement is involved in a wide range of diseases, it is broadly applicable as a clinical indicator.
However, complement can also activate artificially and therefore requires strict control of pre-analytical conditions to ensure accurate measurement. Physicians need to rely on robust, reproducible complement measurements to draw the right conclusions, whether for diagnosis, monitoring treatment response, or assessing disease severity.
Three critical factors for reliable complement measurement are proper sample handling, standardization of methods, and high sensitivity and specificity.
Correct sample handling is essential because the complement system is easily triggered. Blood drawn or processed incorrectly may show false complement activation or degradation. Yet, as Brandwijk et al. showed, nearly half of published studies did not use the proper sample type or handling method. [1] That is a serious problem if clinical decisions depend on the outcome.
Optimal sample handling guidelines:
- Use EDTA-plasma when measuring activation products like C3a, C5a, or sTCC;
- Keep samples on ice;
- Process within 1 hour;
- Store at -80°C;
- Avoid freeze-thaw cycles.
Moreover, standardization is still lacking. There are no universally applied protocols for complement measurement. Laboratories use different antibodies, reagents, calibrators, and detection platforms, leading to inconsistent results across studies or institutions. For diagnostics to be clinically meaningful and comparable, standardized assays and handling protocols must become the norm. This is especially urgent with the growing number of complement-targeted therapies entering the market.
Sensitivity and specificity are also very critical for reliable complement measurement. Complement activation is complex, involving multiple overlapping pathways that can trigger a range of downstream effects. To be clinically useful, assays must have both high sensitivity and high specificity. Sensitivity refers to the ability of a test to detect even very low levels of a specific complement protein or activation fragment. Specificity refers to the ability to detect the correct complement component of a fragment without cross-reactivity to other related proteins.
Without sufficient sensitivity and specificity, complement measurements can easily be misleading, especially given the structural similarities between full protein and their activation products. At Hycult Biotech, we specifically address these challenges. Our assays and antibodies are developed to deliver reliable and reproducible complement protein measurement. We ensure low batch-to-batch variability, have thoroughly validated the specificity of our antibodies and assays, and provide standardized protocols for assay performance. In several cases, we use antibodies that recognize neo-epitopes. Neo-epitopes are specific sites that become exposed only after cleavage of the complement protein. This approach maximizes both sensitivity and specificity, ensuring that our assays detect exactly the right complement molecule.
The expanding role of complement in human pathology continues to innovative therapeutic development across diverse clinical areas. While early successes have validated complement as a druggable system, continued efforts are needed to address the challenges.
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Frequently asked questions
Yes, we can provide you with a poster summarizing the entire pathway. For further reading regarding complement function, regulation and its role in disease, we recommend the following review papers:
Brandwijk Ricardo J. M. G. E. , Michels Marloes A. H. M. , van Rossum Mara , de Nooijer Aline H. , Nilsson Per H. , de Bruin Wieke C. C. , Toonen Erik J. M. ,Pitfalls in complement analysis: A systematic literature review of assessing complement activation, Frontiers in Immunology, Volume 13 – 2022, 2022, 10.3389/fimmu.2022.1007102
Mastellos, D.C., Ricklin, D. & Lambris, J.D. Clinical promise of next-generation complement therapeutics. Nat Rev Drug Discov 18, 707–729 (2019). https://doi.org/10.1038/s41573-019-0031-6
John P. Atkinson, Terry W. Du Clos, Carolyn Mold, Hrishikesh Kulkarni, Dennis Hourcade, Xiaobo Wu, 21 – The Human Complement System: Basic Concepts and Clinical Relevance, Editor(s): Robert R. Rich, Thomas A. Fleisher, William T. Shearer, Harry W. Schroeder, Anthony J. Frew, Cornelia M. Weyand, Clinical Immunology (Fifth Edition), Elsevier, 2019, Pages 299-317.e1, ISBN 9780702068966
Intracellular complement functions as an autonomous regulatory layer that modulates immune cell metabolism, survival, and inflammatory responses, operating inside lysosomes and mitochondria. In contrast, extracellular complement primarily functions in immune surveillance and pathogen elimination via the three pathways in plasma and tissues. A few examples in which intracellular complement differs from extracellular [3]:
| Extracellular | Intracellular |
| Activated in plasma/tissue fluids by classical, lectin, or alternative pathway | Activated within lysosomes, cytosol, or mitochondria |
| Produces C3a/C5a and MAC for immune defence | Produces C3a/C5a to regulate metabolism and inflammation |
| Requires convertase formation for C3/C5 cleavage | May bypass convertases, by using local proteases |
Developing complement inhibitors for clinical use is challenging due to complex pathway crosstalk and activation bypass mechanisms. Non-canonical proteases such as thrombin or kallikrein can cleave C3 or C5 without convertases, allowing bypass activation. This can undermine the efficacy of convertase-targeting therapies [3]. Moreover, functional overlap between pathways enables activation even when one route is blocked. Another challenge lies in the genetic variation in regulatory proteins like factor H and CD46, which can alter disease susceptibility and patient response to therapy. Pathogens further complicate treatment by mimicking host complement regulators, enabling immune evasion. Intracellular complement activity adds complexity and raises questions about how complement inhibitors are expected to work in clinical practice.
Complement dysregulation plays a central role in the development of aHUS and C3 glomerulopathy. In these diseases, uncontrolled alternative pathway activation leads to excessive C3 fragment deposition on host tissue. This results from mutations or autoantibodies affecting regulators such as factor H, factor I, CD46, or factor H-related proteins. In aHUS, factor H variants often cluster in the C-terminal domains responsible for host-surface recognition. This impairs regulation on endothelial cells, promoting thrombotic microangiopathy. In contrast, C3 glomerulopathy-associated variants localize to N-terminal domains affecting fluid-phase C3 regulation. The imbalance causes persistent C3b deposition and complement amplification in the glomerular basement membrane. (The glomerular basement membrane is an extracellular matrix in the kidney.) This drives inflammation, tissue injury, and renal dysfunction. Both diseases reflect distinct but overlapping signatures of alternative pathway dysregulation. Genetic insights from patients have clarified how regulatory defects trigger complement-mediated kidney pathology. [3]
Numerous diseases have been associated with complement system dysfunction. Below is a selection of some widely discussed examples:
- Kidney-Related Diseases (C3G, Lupus Nephritis, aHUS, MN): Characterized by complement dysregulation, immune complex formation, and inflammation, leading to glomerular damage, renal dysfunction, and progressive kidney disease.
- Hematological Disorders (PNH, AIHA, TMA): Conditions involving red blood cell destruction, thrombotic complications, and hemolytic anemia due to impaired complement regulation and immune system dysfunction.
- Neurological Diseases (Multiple Sclerosis, Alzheimer’s Disease, NMOSD, MMN, ALS, Parkinson’s Disease): Chronic neuroinflammatory and neurodegenerative disorders where complement activation contributes to neuronal damage, demyelination, and disease progression.
- Sepsis, SIRS & Ischemia-Reperfusion Injury (IRI): Dysregulated immune activation leading to systemic inflammation, organ dysfunction, and tissue ischemia, relevant in conditions such as stroke, myocardial infarction, and trauma.
- Systemic Autoimmune Disorders (SLE, TMA-related conditions): Autoantibody formation and complement activation drive widespread inflammation, immune complex deposition, and increased disease severity.
- Oncology & Tumor Progression: Complement activation, particularly TCC formation, plays a role in the tumor microenvironment, influencing immune evasion, chronic inflammation, and cancer progression. Complement inhibitors are being explored as potential therapeutic strategies in oncology.
Because of the large number of complement-mediated diseases and recent advancements in large-scale genomics and proteomic research, interest in the complement system has been revitalized, especially as a promising target for therapeutic intervention. This was demonstrated more than a decade ago by eculizumab (Soliris, Alxion), the first complement-specific drug designed to inhibit the key complement component C5.
Therapeutic strategies can target different levels of the complement pathway to prevent or control excessive activation. A central approach is the use of monoclonal antibodies against C5, such as eculizumab, to prevent C5a and membrane attack complex formation. Compstatin analogues act upstream by inhibiting C3 activation, blocking both opsonization and downstream amplification. To target the alternative pathway specifically, inhibitors of factor B or factor D disrupt its amplification loop. Some therapies interfere with convertase assembly or stability, thereby reducing the generation of effector molecules. C5a receptor (C5aR1) antagonists selectively block inflammatory signaling without affecting upstream complement functions. Complement regulation can also be restored by engineered regulators that mimic the activity of proteins like factor H or CD46. These specific targeted deliveries improve a way to modulate complement activity.
The complement system plays an important role in therapeutic research because of its contribution in immune defense, inflammation, and disease pathology. Malfunction of complement activity contributes to autoimmune diseases, inflammatory disorders, neurodegeneration, cancer, and infectious diseases, making it an interesting target for drug development.
Contact us for more information
At Hycult Biotech, we recognize the growing demand for advanced complement research tools that facilitate accurate analysis and targeted intervention in immune-related diseases. Our portfolio includes high-quality complement pathway inhibitors (how to inactivate complement), complement assay kits, and monoclonal antibodies designed to support drug discovery and translational research. On our website, you can search for products that match your research needs. Additionally, we are happy to provide expert advice. Feel free to contact us via the contact form.
[1] Brandwijk RJMGE, Michels MAHM, van Rossum M, de Nooijer AH, Nilsson PH, de Bruin WCC, Toonen EJM. Pitfalls in complement analysis: A systematic literature review of assessing complement activation. Front Immunol. 2022 Oct 18;13:1007102. doi: 10.3389/fimmu.2022.1007102. PMID: 36330514; PMCID: PMC9623276.
[2] Saskia Nugteren, Haiyu Wang, Cees van Kooten, Kyra A. Gelderman, Leendert A. Trouw, Autoantibodies and therapeutic antibodies against complement factor H, Immunology Letters, Volume 274, 2025, 107002, ISSN 0165-2478, https://doi.org/10.1016/j.imlet.2025.107002.
[3] Dimitrios C. Mastellos, George Hajishengallis & John D. Lambris, A guide to complement biology, pathology and therapeutic opportunity, National Center for Scientific Research ‘Demokritos, Volume 24, 2024, 118-141, https://doi.org/10.1038/s41577-023-00926-1