Extracellular and Intracellular Complement Systems

What Is Complement?

The complement system is an important component of the body's intrinsic immunity, consisting of a variety of soluble and membrane-bound proteins, and is the first line of defence against pathogens and participates in the maintenance of the body's dynamic homeostasis. Complement activating factors are intrinsic components of complement, i.e., components involved in the complement activation cascade reaction, including classical complement component 1 (C1)-C9, mannose-binding lectin (MBL) of the lectin pathway, MBL-associated serine protease (MASP), Factor B, Factor H, etc. In the physiological state, these components exist as inactive enzyme precursors. When the complement system is activated, it produces a cascading amplification reaction that ultimately generates the membrane attack complex (MAC), which acts on the cell membrane to form small pores that ultimately lead to the death of the target cell. Regulatory factors refer to a class of proteins, either soluble or in membrane-bound form, that regulate complement activation, e.g., C1-INH, C4BP. These regulatory factors are involved in regulating various aspects of complement activation.

Complement receptors are a class of membrane proteins that are primarily expressed on the membrane surface of immune cells and act by binding to activated complement components. Examples include enhanced recruitment of leukocytes to sites of inflammation, promotion of phagocytosis to remove pathogenic microorganisms, and removal of immune complexes produced at the site of infection or in the blood system. The C5a receptor is a G protein-coupled receptor with high affinity for C5a. C3b and its derivatives related complement receptors include complement receptor type 1 (CR1), CR2, CR3, and CR4. They play a biological role by binding C3b and its degradation products iC3b and C3dg.

Table 1. Complement receptor function associated with C3b and its derivatives

(Source: Mathern DR, et al. 2015)

Intracellular Complement System (ICS)

The intracellular complement system is a relatively independent intracellular immune system that can regulate the function of the corresponding cells, and can be produced by a variety of cell types that can regulate cellular function within the cell. Unlike the extracellular complement system, which is traditionally synthesized and secreted by hepatocytes, ICS has been found in a wide range of cells other than hepatocytes, with intracellular complement components such as C3, C5, factor H (FH) and their corresponding receptors. The ICS is also named the composome because of its close association with the formation of nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammatory vesicles.

Figure 2. Noncanonical functions of intracellular complement
(Source: West EE, et al. 2018)

Complement C3

Intracellular C3 is mainly derived from receptor-mediated internalization of extracellular C3 hydrolysis products and self-synthesis of C3 proteins. Spontaneous hydrolysis of thioester bonds by C3 in the peripheral circulatory system occurs at a slow rate to form a C3 hydrolysis product, which enters the cytosol by binding to fibroblast lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP/α2MR) and localizes to act in early endosomes. Cells can synthesize C3 proteins on their own, and some investigators have found that a signaling pathway mediated by the binding of human T-cell integrin lymphocyte function-associated antigen 1 (LFA-1) to endothelial cell-expressed intercellular adhesion molecule 1 (ICAM-1) induces T-cell C3 protein expression via activator protein 1 (AP-1).

Intracellular C3 plays an important role in maintaining CD4⁺ T cell homeostasis. Cellular homeostasis is maintained within resting T cells by maintaining basic cellular glycolytic metabolism, and when T cells are activated, C3b binds to CD46, which contains the functional end of the cytoplasmic terminus of CYT-1 on the surface of the T cell, and promotes differentiation of type I helper T (Th1) cells. When the T-cell antigen receptor (TCR) recognizes antigen and is activated, intracellular stores of C3aR and CD46 are rapidly translocated to the cell surface, while C3a and C3b are translocated extracellularly to bind to cell surface C3aR and CD46, respectively. Subsequent C3b-CD46 signaling drives GLUT-1 and L-type amino acid transporter 1 (LAT-1) expression and mediates upregulation of LAMTOR5 expression. Eventually large amounts of glucose and amino acids that are in-fluxed into the cell work together with LAMTOR5 to promote mTORC1 activation, causing γ-interferon (IFN-γ) secretion and Th1 cell formation.

Intracellular C3b is also involved in the cytotoxic effects of CD8⁺ T cells. When the organism is stimulated by relevant pathogens, CTSL rapidly lysis C3 to generate active C3b, which binds to CD46 on the cell surface via autocrine secretion and drives higher levels of fatty acid synthase, fatty acid-binding protein 5 expression, which in turn promotes the secretion of IFN-γ and granzyme B and induces cytotoxicity production. In addition, intracellular C3 is thought to be closely related to cancer development, and the increase in glycolysis resulting from its activation may be one of the mechanisms of tumorigenesis.

Complement C5

C5 is present in a variety of cells, but its origin is unclear. Similar to intracellular C3, activation of intracellular C5 in CD4⁺ T cells is also involved in Th1 cell formation. In conjunction with TCR and CD46, intracellular C5 is lysed into C5a and C5b, and C5a binds to C5aR1, which is only expressed intracellularly, inducing ROS generation. The production of endogenous ROS promotes the assembly of NLRP3 inflammatory vesicles and the secretion of IL-1β, which in turn maintains the Th1 response. When the activation signal is terminated, another C5a receptor, C5aR2, can form a dimer with C5aR1, thereby inhibiting the C5aR1-mediated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and suppressing the Th1 effect, and the T-cells are then shifted from activated to resting state.

The function of the C5 system varies depending on the cell type. For example, in CD8⁺ T cells NLRP3 expression is not required for normal IFN-γ secretion and CTL activity, and in macrophages C5a-mediated signaling pathways even inhibit NLRP3 assembly and activation.

Complement ELISA Kits

Complement Antigens

Complement Antibodies