24 Laboratory and clinical assessment of the complement system

Lothar Thomas

The complement system provides innate defense against microbial pathogens and is a "complement" to antibody-mediated immunity /1, 2/. The complement cascade is an enzymatic reaction cascade involving the classical pathway (CP), alternative pathway (AP), the mannan binding lectin pathway (MBL), and the activation by plasmin an kallikrein (Fig. 24-1 – Pathways activating the complement system).

The complement system has pro inflammatory activity and works in part by a cascade involving limited proteolysis whereby one component activates the next, resulting in a strong amplification. The overall goal is deposition of complement fragments on pathogens for the purpose of opsonization, lysis and liberation of peptides that promote the inflammatory response.

The complement system consists of more than 30 proteins in plasma and at least 9 membrane proteins (Tab. 24-1 –Proteins of the complement system). Approximately 90% of the quantity of plasma complement proteins is synthesized by the liver and the vast majority reside in the blood. Mucosal secretions and tissues contain 5-10% of the corresponding concentrations in serum. However, inflammation at these sites increases complement concentration by increased diffusion and local synthesis of the relevant proteins. Cytokines of the acute-phase response (TNF, IL-1, IL-6) can increase the hepatic synthesis of complement components several folds in hepatocytes.

Deficiency states of plasma complement components may be acquired or inherited /1, 2/. Acquired deficiencies are relatively common, may be of acute or chronic etiology, and are frequently reversible with treatment of the disorder responsible for the deficiency. Inherited defects of individual complement proteins are uncommon in the general population. Defects of complement components predispose to infections and autoimmune diseases. Even though total deficiency of a complement component is rare, patients presenting with certain bacterial infections and autoimmune disorders.

24.1 Indication

Infections, especially with Neisseria sp., S. pneumoniae, Haemophilus influenzae type b

Autoimmune disease:

• Systemic lupus erythematosus (SLE), generalized vasculitis, glomerulonephritis

Hemolytic disease

Cryoglobulinemia

Suspected hereditary angioneurotic edema.

24.2 Method of determination

The functional activities of the classical (CH₅₀) and of the alternative pathway (AP₅₀) can be determined. Both assays provide approximately the same amount of information on the C5b-C9 membrane attack complex /3/. The patient serum is the complement source, and sheep erythrocytes are used as the indicator system. Human serum is used as control (normal), and inactivated patient serum as blank (inactivation at 56 °C for 30 min.). The number 50 results from the serum dilution at which 50% hemolysis occurred.

CH₅₀ and AP₅₀ are used to screen for complement deficiency. Because both tests include the 6 terminal components (C3, C5, C6, C7, C8, and C9), the results will be low for both tests, if one or more of these components are missing. If a component of the classic pathway is missing, the CH₅₀ will be low or absent, but the AP₅₀ will be normal, whereas if a component of the alternative pathway is low or missing, the reverse will be true /2/.

24.2.1 CH₅₀ assay

Principle: a definite volume of antibody coated sheep erythrocytes is incubated with the complement source (serum) to be analyzed in geometric dilution. Due to the activation of C3 convertase and the membrane attack complex, the erythrocytes are hemolyzed. Subsequently, the reagent mixtures are centrifuged and the degree of hemolysis is determined photometrically by measuring the hemoglobin concentration in the supernatant. In a semi logarithmic graph, the reciprocal value of the serum dilution (ordinate) is plotted against the absorption (abscissa). The unit definition is method dependent.

The formation of the membrane attack complex (MAC) on the cell requires the sequential action of all 9 components of the classical (C1, C4, and C2) and terminal (C3, C5, C6, C7, C8, and C9) pathways.

Principle of complement determination by haptenized liposomes: liposomes are haptenized on their surface with dinitrophenol. Liposomes, the complement source (serum), antibodies directed against dinitrophenol, and glucose-6-phosphate (GP) are incubated. The liposomes, containing the enzyme glucose-6-phosphate dehydrogenase (G6PDH), are lysed by the antibody bound complement and the intrinsic G6PDH is released. The G6PDH catalyzes the transformation of GP to ß-gluconolacton and reduces NAD to NADH. The increase in NADH is proportional to the activity of complement.

Der CH₅₀ assay and the liposome assay are the best screen for complement deficiency. The absence or decrease of activity implies that at least one of the components is missing or low.

24.2.2 AP₅₀ assay

Principle: the assay is performed like the CH₅₀ assay, except that sensitized rabbit erythrocytes are used and the formation of classical C3 convertase is prevented. This activation pathway is Ca²⁺-dependent and is inhibited if the incubation reagents of the AP₅₀ assay do not contain any Ca²⁺.

The AP₅₀ assay depends on the sequential activation of factors D, B, P, C3, C5, C6, C7, C8, and C9.

As with the determination of individual coagulation factors, the activity analysis of individual complement components can be performed using deficient plasma (i.e., plasma in which the complement component to be analyzed is absent). For this, constant volumes of deficient plasma are incubated with increasing volumes of patient plasma.

24.2.3 Complement activation enzyme immunoassay (CAE)

Principle: the classical complement activity is determined using a microtiter plate in which the wells are coated with immune complexes. After the patient’s sample is added, C1q contained in the sample is bound to the immune complexes and the complement system is activated, leading to the production of C9. The concentration of C9 is measured using a monoclonal, enzyme labeled anti-C9 antibody /4/.

24.2.4 Immunochemical determination

Principle: the concentration of individual complement components is measured by immunonephelometric, and immunoturbidimetric assays. Using commercially available reagents, C3 or C3c, C4, C1q, C1-esterase inhibitor (C1-INH), and factor B (C3 activator) can be routinely measured.

C3c is a stable C3 fragment which is formed as a result of factor I acting on the unstable C3b. The concentration of C3c is an indicator of C3 turnover and generated within hours of blood collection.

Because many of the complement components are acute phase proteins decreases due to activation might be masked by increases in the synthesis during an inflammatory episode.

Quantitative determination of complement component split products can be used to determine the pathway of activation:

• Markers for activation of the classic pathway and the mannan binding lectin pathway are C4a and C4d
• Marker for activation of the alternative pathway is Bb
• Markers for activation of the terminal pathway are C3a, iC3b and C5a.

24.2.5 C1 inhibitor (C1-INH) activity

C1q and C1-INH autoantibody action can be determined by using ELISA or spectrophotometric assays /5/.

Enzyme immunoassay: the patient’s sample is preincubated with biotinylated C1s. The resulting C1-INH-C1s complex is bound to avidin coated micro titer wells. The C1-INH is then detected with a horseradish peroxidase linked antihuman C1-INH antibody.

Spectrophotometric assay: the patient’s sample is incubated with excessive amounts of C1-esterase inhibitor and the substrate methyloxycarbonyl-L-lysyl-(ε-carbobenzoxy)-glycyl-L-arginyl-p-nitroaniline. The amount of cleaved p-nitroaniline is inversely proportional to the concentration of C1-INH in the sample.

24.2.6 Determination of cell bound complement components

Direct antiglobulin test (Coombs test): anti erythrocytic antibodies bind complement, especially C3, to the surface of erythrocytes and may leave it behind when they are split off. Such behavior is displayed, in particular, by erythrocyte bound IgM antibodies. Accordingly, in the direct Coombs test, detection of erythrocyte bound C3 in the absence of IgM is typical of cold agglutinins.

24.2.7 Determination of complement receptors

The determination of the complement receptors CR1 (CD35), CR2 (CD21), CR3α-chain (CD11b) and CR3β-chain (CD18) on blood cells is performed using fluorescence labeled monoclonal antibodies. Indirect immunofluorescence or flow cytometry is employed.

24.3 Specimen

EDTA plasma (until the separation of plasma, blood is stored at 4–8 °C): 3 mL

Serum is used for determining the protein concentrations of C3c, C1q and C1-INH: 1 mL

24.4 Reference interval

Refer to Ref. /3, 4, 6/ and Tab. 24-2 – Reference intervals of complement investigations.

24.5 Clinical significance

Awareness of complement deficiency states as a cause of disease is not strongly established within health care. Hereditary angioedema, systemic lupus erythematosus (SLE) and recurrent meningococcal disease are probably the most common clinical settings in which complement deficiency tends to be suspected, even if this is not always the case /7/.

Investigations that are routinely performed for evaluation of the complement system include determination of CH₅₀ or CAE and/or measurement of the protein concentrations of C3 and/or C3c, C4, factor B, and C1-INH.

24.5.1 Genetic complement deficiency diseases

Most complement components are inherited in an autosomal co dominant pattern. Typical components of this type include C1-Inh, C2, C3, C5, C6, C7, and C9 /2/. The subcomponents C1q, C1r and C1s are required for C1 function. Deficiencies of the subcomponents and of C4 and C2 are all associated with an increased risk of pyogenic infection.

Children with C2 deficiency may experience invasive infections with S. pneumonia and H. influenza type B that cease during adolescence suggesting establishment of acquired immunity. In C2 deficiency, anti capsular IgM and IgG antibodies might trigger immune adherence of S. pneumonia to erythrocyte complement receptor CR1 by recruitment of C4 /8/.

Recurrent invasive infections caused by Neisseria meningitidis or Neisseria gonorrhoea occurs in the setting of a deficiency of a MAC component (C5, C6, C7, or C8) or of the alternative pathway component properdin /2/.

The evidence of SLE in patients with C1q, C4 or C2 deficiency is 90%, 75% and approximately 15%, respectively /9/.

Reduced C4 and C3 levels in the setting of lupus nephritis are an important predictor of more severe disease and poor outcome. Total deficiency of C3 is associated with development of membranoproliferative glomerulonephritis /10/.

Persons with deficiencies of the classical pathway or C3 show reduced antibody responses to thymus-dependent antigens and impaired IgM/IgG switching /11/. The effect is due to the C3 derived fragment C3d, a specific ligand of complement receptor 2 (CR2) that was shown to have strong dose-dependent adjuvant effect.

Factor D deficiency and the X-linked properdin deficiency result in the selective impairment of alternative pathway function. The far most common pathogen encountered is Neisseria meningitidis.

Refer to:

• Tab. 24-3 –Genetic complement deficiency diseases
• Tab. 24-4 –Hereditary deficiency of complement components

24.5.2 C1 inhibitor (C1-INH) deficiency

C1-INH is a glycoprotein of the serine protease inhibitor family (serpines) and synthesized in the hepatocytes.

C1-INH controls /12/:

• The spontaneous auto activation of C1 and of activated C1. Functional C1-INH deficiency leads to activation of the classical pathway and to reduced levels of C4 in serum.
• The activated proteases of the contact phase of the coagulation system, including Hageman factor (F XIIa), prekallikrein, F XI, and high-molecular-weight kininogen.

Refer to Fig. 24-2 – The role of C1-INH in the regulation of fibrinolysis, the classical pathway of the complement system, and the bradykinin system.

24.5.2.1 Hereditary angioedema

Hereditary angioedema (HAE) is a rare genetic disorder characterized by recurrent episodes of non painful, non pruritic, and non-erythematous subcutaneous and submucosal swelling that subsides in 48–74 hours. These angioedema attacks result from uncontrolled activity of contact system components factor XIIa and plasma kallikrein, which leads to excessive bradikinin release and subsequent locally increased vascular permeability. The majority of angioedema cases are either decreased levels (type 1) or reduced functionality (type II) of C1 esterase inhibitor. Both types are clinically indistinguishable.

C1-INH is the main inhibitor of kallikrein and plasma F XIIa and thus an important regulator of the activation of the kallikrein-kinin system. During an acute HAE attack, kallikrein is not sufficiently inhibited by C1-INH. As a result, the kallikrein-kinin system is activated and there is increased production of bradykinin, which increases vascular permeability and thus promotes the development of edema.

C1-INH deficiency leads to uncontrolled activation of the classical complement pathway. This in turn leads to increased consumption of C2 and C4, but no effective C3 convertase is produced, and therefore no C3 is consumed.

If C4 deficiency is not present as a result of other causes, a decrease in C4 in combination with normal C3 levels may indicate C1-INH deficiency.

The criteria indicating congenital or acquired C1-INH deficiency are a C1-INH activity of ≤ 25% of normal in the presence of the clinical symptoms described. However, activity of C1-INH can be very variable, depending on the genetic status. An explanation for this is the presence of C1s-C1r-C1-INH complexes or of anti-C1-INH autoantibodies. Findings in C1-INH deficiency are shown in Tab. 24-3 – Genetic complement deficiency states.

24.5.2.2 Kidney damage

Development of kidney disease in the presence of autoimmune disease occurs by several mechanism. Refer to Tab. 24-3 –Genetic complement deficiency states.

24.5.2.3 Acquired complement deficiency diseases

Autoimmune diseases, especially those featuring immune complexes, often result in secondary complement deficiency because complement activation outstrips hepatic synthesis.

Clinical manifestations and pathophysiology of acquired complement deficiency diseases are /2/

• Immune complex deposition. Immune complexes that are not properly eliminated can cause inflammation. Serum sickness (acute immune complex disease) is caused by the presence of a relatively large amount of foreign antigen to which the host mounts an immune response. Mixed cryoglobulinemia is often caused by the immune response to hepatitis C virus or by complexes with rheumatoid factor.
• Autoantibody syndrome. In type 2 hypersensitivity reactions, the autoantibody binds to a fixed antigen on cells or tissue sites and activates complement. C3 nephritic factor is an autoantibody against the alternative pathway convertase. It results in secondary C3 deficiency.
• Paroxysmal nocturnal hemoglobinuria (PNH). The PNH erythrocytes are highly susceptible to complement mediated lysis because of absence of two complement regulators, decay accelerating factor (CD55) and CD59 the inhibitor of the membrane attack complex (MAC)
• Ischemia re perfusion injury. Perfusion of ischemic tissue is associated with marked inflammatory response that might lead to further undesirable tissue and organ damage. The major mediators of damage are the anaphylatoxins C3a and C5a that attract neutrophils that in turn release enzymes and other mediators and also generate oxygen radicals.

Refer to:

• Tab. 24-4 –Hereditary deficiency of complement components
• Tab. 24-5 –Acquired deficiency of complement components
• Tab. 24-6 – Acquired (secondary) complement deficiency characterized by immune complexes
• Tab. 24-7 –Interpretation of CH₅₀ and AP₅₀
• Tab. 24-8 –Interpretation of C3 and C4
• Tab. 24-9 –Activation pathway in the presence of hypocomplementemia

24.5.2.4 Hypercomplementemia

Many complement components, especially C3, C4 and C1-INH, are acute-phase proteins. During an acute-phase response, they are synthesized not only in the liver but also in macrophages. Major causes of elevated complement concentrations include systemic infectious diseases, noninfectious chronic inflammatory conditions such as rheumatoid arthritis, and physiological conditions such as pregnancy. The detection of elevated complement levels is of little value for the diagnosis of such diseases and conditions. However, increased synthesis of complement components must be suspected if, in the setting of active immune complex disease, activation of complement and thus a reduction in CH₅₀ or in the concentration of complement components is expected but does not occur, and both a normal CH₅₀ activity and normal complement component concentration is measured.

The determination of C-reactive protein (CRP) can be useful. If CRP is elevated, an acute-phase response is present and the complement activity or complement concentration is not generally an indicator of an immune complex disease.

The complement component C5a has a critical role in sepsis. In diffuse complement activation, such as in sepsis, high concentrations of C5a inhibit the activity of polymorphonuclear granulocytes by down regulating C5a receptors of these cells and thus making them unable to respond adequately to C5a. Neutralization of C5a by monoclonal antibodies improves sepsis /13/.

24.6 Comments and problems

Blood sampling

EDTA (1.5–2.0 mg/mL whole blood) should be used as anticoagulant for the determination of functional complement activity and C1-INH and for the immunochemical determination of C3 and C4. During the coagulation process, C1-INH is consumed due to the activation of serine proteases. Since complement activation does not occur in EDTA plasma, C4 is more stable in plasma than in serum. Plasma must be separated from erythrocytes within 1 h in order to prevent in vitro activation of the complement system /12/.

In whole blood and serum C3 is transformed into the functionally inactive fragments C3c and C3dg which may cross react with the antibodies used in the immunochemical assays. The determination of C3 should be performed in EDTA plasma whereas C3c should be determined in serum, but not until 24–48 h after blood collection, or after incubation at 37 °C for 1 h, because then all the C3 will have been transformed into C3c.

The classical complement pathway is activated in vitro in serum or heparinized plasma after storage at low temperature. As a result, CH₅₀ and C4 levels are reduced. No change in levels occurs in EDTA plasma (cold or at 37 °C) or in serum kept at 37 °C until the time of analysis. Cold dependent complement activation occurs in samples from patients with SLE, chronic liver and kidney diseases, and in 41–89% of samples from patients with hepatitis C infection /14/.

Method of determination

The CH₅₀ test result is considered pathologic only if there is a marked decrease in classical pathway complement activity or a greater than 50% reduction of a complement component. This is based on the fact that there is normally significant excess of complement.

In immunonephelometric and immunoturbidimetric assays for C3 it is important to pay attention to the fact that the reference material used for standardization (e.g., the reference preparation for human serum proteins RPPHS/CRM470) contains C3c. C3c, which is detected by commercial assays, is generated by C3 cleavage several hours after blood collection. If assayed shortly after blood collection, C3c will be underestimated, since the C3 to C3c conversion is not yet complete. Therefore, due to the presence of remaining intact C3, concentrations measured in fresh patient serum are lower compared to those measured in old serum.

Stability

For the CH₅₀ assay, plasma is stored at room temperature if the determination is performed on the same day; otherwise storage at –20 °C for several days or at –70 °C for prolonged periods of time. For determination of protein concentration (e.g., of C3c) serum can be mailed.

24.7 Pathophysiology

The complement system:

• Consists of approximately 30 proteins, excluding cell surface receptors and control proteins, and makes up 15% of the serum globulin fraction
• Is a component of innate immunity (see Section 21.1.4 – Innate immune response) and an effector of antibody mediated immune defense.
• Has highly conserved structures that allow it to recognize molecular antigen patterns and eliminate the pathogens
• Is activated through three pathways. After activation, the complement components interact with each other in a sequential way to produce effector molecules. This sequence of interactions is called the complement pathway. The system exerts its effects and controls itself via functional units (Fig. 24-3 – Clinical pathway, lectin pathway and alternative pathway) /1/.

The complement system involves three pathways.

• The classical pathway (CP) of activation with the components C1, C2, C3, C4. The CP is activated by natural IgM antibodies, which recognize a certain molecular antigen pattern, or by IgG antibodies produced shortly after contact with a pathogen
• The alternative pathway (AP) of activation with the complement components C3, factor B, factor D and properdin. The alternative pathway directly recognizes molecular antigen patterns on bacteria, fungi or injured body cells
• The mannan binding lectin pathway (LP). The lectin pathway recognizes foreign carbohydrate structures and recruits specific serine proteases to activate complement component C4.

All of the activating pathways converge to form C3 convertases. Cleavage of C3 yields C3a and C3b, the latter which triggers the formation of C5 convertase. The C5 convertase cleaves C5 into C5a and C5b, the latter oligomerizes with C6, C7, C8 and multiple C9 molecules to form the membrane attack complex (MAC), which causes direct destruction of the pathogen /34/.

The components of the complement system are activated through proteolysis, which results in the formation of new components. N-terminal proteolysis of C3, C4 and C5 yields peptides, which cause:

• Chemotaxis by binding to receptors of inflammatory cells
• The release of enzymes stored in the granules of inflammatory cells
• The synthesis of inflammatory cytokins
• A change in vascular permeability by binding to the receptors of vascular cells.

The endpoints of complement functions are promotion of inflammation, enhancement of the immune response, and elimination of pathogens.

Because actions of the complement system can have deleterious effects on the host active control occurs through the action of inhibitors and control proteins e.g., factor H, factor I. Refer to:

• Tab. 24-10 – Regulation of the complement system by control proteins

The cell membrane bound complement receptors (CR) mediate the binding of complement containing immune complexes to cells. Membrane bound immune complexes are more rapidly phagocytized and better removed so that they cannot precipitate in the circulation and induce immune complex disease (serum sickness).

Activation of the classical pathway (CP)

The CP is activated by immune complexes which contain IgM, IgG₁, IgG₂ or IgG₃ as well as by proteolytic enzymes, heparin, and viruses /1, 2, 15/. The activation sequentially causes the proteolytic cleavage of the complement components C1, C4, and C2 with formation of C3 convertase.

• Activation usually begins when the tulip shaped C1q binds to the Fc portion of an antibody in an immune complex. The subcomponent C1r then auto actives and cleaves C1s. C1s in turn cleaves C4 and C2.
• Cleavage fragments of C4 and C2 assemble on the target to form C3 convertase (C4b2a), which in turn cleaves C3 to produce C3a and C3b. The half life of C3 convertase is approximately 3 min.
• C3b deposits on the target where it serves as an opsonin and interacts with C4b2a to form the C5 convertase (C4b2a3b)
• The C5b formed by the C5 convertase initiates the terminal lytic sequence C5 through C9 by binding C6 and C7. This complex attaches to the cell membrane and subsequently engages C8 and multiple C9s to form the membrane attack complex (MAG).

Refer to

• Fig. 24-4 – Activation of the classical complement pathway

The alternative pathway of the complement system can be activated by aggregated immunoglobulins, fungi, bacteria, viruses, polysaccharides and self initiation /2/. This pathway is continuously turning over on a small scale.

• Attachment of active C3 on a surface lacking complement regulators permits this pathway to rapidly amplify via a feedback loop.
• The C3b generated forms convertases and thus gives more rise to more C3b.
• The C3 convertase is initiated when the proenzyme, factor B, attaches the target bound C3b.
• Factor B than undergoes cleavage by factor D to produce the alternative convertase (C3bBb). Properdin stabilizes this complex
• As more C3 is cleaved by the convertase to C3bBb, an amplification loop is set up that permits large amounts of C3b to be deposited on the target. Regardless of how the complement cascade is initiated the alternative pathway accounts for more than 75% of complement activation products. This because the alternative pathway C3 convertase (C3Bb) not only triggers the C5 convertase; it also cleaves more C3 molecules leading to the rapid generation of C3b resulting in an amplification loop /34/.
• The C5b formed by the C5 convertase (C3bBb3b) initiates the terminal lytic sequence C5 through C9 by binding C6 and C7. This complex attaches to the cell membrane and subsequently engages C8 and multiple C9s to form the membrane attack complex (MAG).

Refer to Fig. 24-5 – Activation of the alternative complement pathway

Animal lectins such as mannose binding protein or mannan binding protein (MBP) recognize mannose or N-acetylglucosamine on the surface of bacteria. MBP is a member of the collectin family, a group of Ca²⁺-dependent lectins /16/. The structure and function of collectins resemble those of C1q. MBP binds sugar residues on the microbial surface and the mannose associated serine protease (MASP), analogous to C1r and C1s, cleave C4 and C2. From that point, C4b and C2a form the C3 convertase, and activation proceeds to the terminal C5-C9 /2/.

Refer to Fig. 24-3 – Classical pathway, lectin pathway and alternative pathway

Biological functions of the complement system

The main biological functions of the complement system are /1, 6, 17/:

• Opsonization and phagocytosis by binding of C3b to target cells. Immune complex linked C3b binds to specific C3b receptors on macrophages and granulocytes, thus facilitating, for example, the phagocytosis and proteolytic degradation of infectious pathogens coated with antibodies
• Triggering of an inflammatory response by producing the anaphylatoxins C3a and C5a and activation of chemotaxis (C5a) of inflammatory cells. The anaphylatoxins stimulate the release of histamine from mast cells. Histamine causes increased vascular permeability by inducing contraction of the smooth muscles of the blood vessels. As result of this, more complement components, antibodies and inflammatory cells can migrate into the extravascular compartment.
• Insertion of the membrane attack complex into the cell membrane of pathogenic cells and bacteria. This leads to osmotic lysis of the target cells through the formation of a transmembrane channel.
• Acceleration of the immune response by binding of C3 fragments to target cells.
• Removal of immune complexes from cell surfaces by solubilization of large immune aggregates, inhibition of IC formation, and binding of immune complexes to macrophages, which then phagocytize the immune complexes.
• Removal of apoptotic cells via the classical pathway by the binding of C1q to the cell surface
• Release of neutrophil granulocytes from the storage pool of bone marrow by C1s and C3d kallikrein complexes. In addition, C1s activates platelets, the blood coagulation, the fibrinolytic system, and the kinin system.

The synthesis of complement components occurs in the liver and in monocytes/macrophages at the site of inflammation. Complement components are secreted in an inactive state. In healthy people, low grade activation of the complement system occurs continuously (activation at rest) since factor D, which is the only protein secreted in an active state, continuously converts C3 into the activated form C3b. Therefore, the daily turnover of C3 is about one half of total body C3. The plasma concentration of the complement components is 3–4 g/L, of which C3 accounts for about 30%.

Fig. 24-6– Regulation of the complement pathways by control proteins

Complement receptors

Because of their complement receptors, macrophages and granulocytes bind complement fragments and are involved in the defense against pathogens or elimination of immune complexes /16/. Complement receptors also serve as binding sites for complement fragments that are produced during activation at rest and inactivated by factor I (Tab. 24-11 – Control proteins on the cell membrane of blood cells).

The receptor CR1 is a glycoprotein which is located on neutrophils, eosinophils, erythrocytes, monocytes, dendritic cells and certain T and B cells. CR1 binds C3b, C4b and particles which are coated with these fragments. This complement receptor is known:

• To bind C3b, which is produced during low grade activation, in order to be transformed into C3d and C3dg by factor I
• To facilitate the phagocytosis of immune complexes and particles coated with C3b and C4b
• To directly participate in adaptive immune defense, since C3b and C4b binding results in the activation of macrophages which release interleukin-1. The latter then activates T cells.

The receptor CR2 is located on B cells and follicular dendritic cells of lymphatic organs and binds C3d and Epstein-Barr virus. The binding of C3d results in the proliferation of B cells.

The CR3 receptor belongs to the adhesion proteins and is located on neutrophil and eosinophil granulocytes as well as monocytes. This receptor binds C3bi, which is C3b that has been inactivated by factor I and displays no in vitro hemolytic activity. Granulocytes and monocytes are activated to phagocytize particles coated with C3bi.

The fragments generated during complement activation play an important role in the elimination of immune complexes. In the body, antigen-antibody reactions are an ongoing process which is associated with formation of immune complexes of a size that can result in their precipitation. If these complexes are not removed, they precipitate in the tissues and thus cause inflammatory response. Attachment of complement fragments to the immune complexes causes these complexes to be labeled and to be bound by the complement receptors (CR) of the blood cells. CR1 carrying cells, for example, transport immune complexes to the reticuloendothelial system in the liver and spleen and thereby contribute to their elimination.

In complement component deficiencies, elimination of immune complexes is impaired, and often an association with immune complex diseases and autoimmunopathies can be diagnosed.

Products of complement activation

As a result of complement activation, biologically active cleavage products, or fragments, are produced from the inactive complement components. These products include C3a and C5a, also known as anaphylatoxins, as well as C3e, whose function is largely unknown.

C3a has a molecular weight of 9.1 kDa and induces:

• Degranulation of mast cells and basophilic granulocytes with a release of histamine
• Release of interleukin-1 from monocytes
• Contraction of smooth muscles
• An increase in vascular permeability
• Suppression of antibody response.

C5a has a molecular weight of 11.2 kDa and induces:

• Chemotaxis of neutrophil and eosinophil granulocytes as well as monocytes. After binding of C5a to special cell receptors, these cells are stimulated and migrate into the inflammatory area.
• Release of interleukin-1 from monocytes
• Release of histamine from mast cells
• Contraction of smooth muscle cells and increase in vascular permeability.

C4a has a molecular weight of 9 kDa and properties similar to C3a and C5a.

C3e has a molecular weight of 10 kDa and increases the permeability of cutaneous blood vessels for mediator proteins during inflammation, mobilizes leukocytes from the bone marrow, and activates granulocytes.

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