24

Laboratory and clinical assessment of the complement system

24

Laboratory and clinical assessment of the complement system

24

Laboratory and clinical assessment of the complement system

24

Laboratory and clinical assessment of the complement system

  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 /12/. 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 /12/. 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. /346/ 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:

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 characterized by recurrent episodes of non painful, non pruritic, and non-erythematous subcutaneous and submucosal swelling that subsides in 48–74 hours. It is not the inhibitory effect of C1-INH on the complement system but its effect on the kallikrein-kinin system that is crucial in triggering HAE, because 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 indicates 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. A distinction is made between:

  • Hereditary angioedema (HAE), which is due to impaired synthesis of C1-INH
  • Acquired angioedema (AAE), which is due to increased catabolism of C1-INH

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:

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:

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 /1215/. 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

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 /1617/:

  • 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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20. Moulds JM, Warner NB, Arnett FC. Complement component 4A and 4B levels in systemic lupus erythematosus: quantitation in relation to C4 null status and disease activity. J Rheumatol 1993; 20: 443–7.

21. Zadura AF, Theander E, Blom AM, Trouw LA. Complement inhibitor C4b-binding protein in primary Sjögren´s syndrome and its association with other disease markers. Scand J Immunol 2009; 69: 374–80.

22. Nangaku M. Complement regulatory proteins in glomerular diseases. Kidney Int 1998; 54: 1419–28.

23. Wagner M, Vorlaender KO. Methodenvergleiche zur Bestimmung der hämatologischen Komplementaktivierung in der Routinediagnostik. Ärztl Lab 1985; 31: 265–72.

24. Frank MM. Complement deficiencies. Pediatr Clin North Am 2000; 47: 1339–53.

25. Jarvis JN, Pousak T, Krenz M, Jobidze M, Taylor H. Complement activation and immune complexes in juvenile rheumatoid arthritis. J Rheumatol 1993; 20: 114–7.

26. Homann C, Varmig K, Hogasen K, Mollnes TE, Graudal N, Thomsen AC, Garred P. Acquired C3 deficiency in patients with alcoholic cirrhosis predisposes to infection and increased mortality. Gut 1997; 40: 544–9.

27. Unsworth DJ. Complement deficiency and disease. J Clin Pathol 2008; 61: 1013–7.

28. Wyatt RJ, Julian BA, Rivas ML. Role for specific complement phenotypes and deficiencies in the clinical expression of IgA nephropathy. Am J Med Sci 1991; 301: 115–23.

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30. Busse PJ, Christiansen SC. Hereditary angioedema. N Engl J Med 2020; 382 (12): 1136–48.

31. Hahn J, Hoffmann TK, Bock B, Nordmann-Kleiner M, Trainotti S, Greve J. Angiooedema – aninterdisciplinary emergency. Dtsch Arztebl Int 2017; 114: 489–96.

32. Whaley K, Sim RR, He S. Autoimmune C1-inhibitor deficiency. Clin Exp Immunol 1996; 106: 423–6.

33. Levy Y, George J, Yona E, Shoenfeld Y. Partial lipodystrophy, mesangiocapillary glomerulonephritis, and complement dysregulation: an autoimmune phenomenon. Immunoll Res 1998; 18: 55–60.

34. Brodsky RA. Complement in hemolytic anemia. Blood 2015; 126: 2459–65.

35. Noris M, Ruggenenti P, Perna A, Orisio S, Caprioli J, Skerka C, et al. Hypocomplementemia discloses genetic predisposition to hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: role of factor H abnormalities. J Am Soc Nephrol 1999; 10: 281–93.

36. Homann C, Varmig K, Hogasen K, Mollnes TE, Graudal N, Thomsen AC, Garred P. Acquired C3 deficiency in patients with alcoholic cirrhosis predisposes to infection and increased mortality. Gut 1997; 40: 544–9.

37. Marschang P, Ebenbichler CF, Dierich MP. HIV and complement: role of the complement system in HIV infection. Int Arch Allergy Immunol 1994; 103: 113–7.

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40. Eisele B, Delvos U. From localized angioedema to generalized capillary leak syndrome: evidence for a pivotal role of C1-inhibitor in septic shock-like syndromes. In: Reinhart K, Eyrich K, Sprung C, eds. Update in intensive care medicine 18: sepsis. Heidelberg: Springer, 1994: 501.

Table 24-1 Components of the complement system /1/

Compo-
nent

MW

(kDa)

(mg/L)

Function

Classical pathway

C1q

400

70

Binding to active surfaces and formation of activated C1s, which cleaves C4 and C2

C1r

190

35

C1s

85

30

C4

205

600

Part of C5 convertase (Fig. 24-1 – Pathways activating the complement system), is cleaved to generate the anaphylatoxin C4a

C2

117

25

C2a is the catalytic fragment of C5 convertase

Alternative pathway

C3

185

1.300

The C3b fragment binds to activating surfaces and is part of C5 convertase; the C3a fragment is an anaphylatoxin. C3bi is C3b inactivated by factor I.

Factor B

95

20

Part of C5 convertase

Factor D

23.5

1.8

Activates C3 to C3b. Factor D is the only complement component that circulates in an active form. Cleaves factor B.

Membrane attack complex C5b-C9

C5

190

160

The C5b fragment is the initiating component of the membrane attack complex (MAC)

C6

120

65

Component of the MAC

C7

110

55

Anchors the C5b-C7 complex to the cell membrane

C8

155

55

Binds to C5b-C7 to form a transmembrane channel

C9

79

200

Polymerizes in order to enlarge the transmembrane channel.

Regulatory proteins

Refer to

C1-INH

105

180

Inhibits the synthesis and proteolytic activity of C1q and C1r. Inhibits plasma kallikrein and F XIIa.

C4bp

> 500

300

Regulates complement activation of the classical pathway in the fluid phase. Binds to C4, is a cofactor to factor I, both jointly degrade the classical C3 convertase, C4b2a

CD59

20

Known as membrane inhibitor of reactive lysis (MIRL), as homologous restriction factor 20 (HRF-20), or protectin. Binds the α-unit of C8 and the C9b domain and prevents formation of the membrane attack complex by inhibiting the incorporation of C8 and C9.

CR1

The Complement receptor 1 (CR1) is a cofactor to factor 1 and inhibits the activation of the classical and alternative C3 convertases

DAF

83

Decay accelerating factor (DAF, CD55) inhibits the formation and accelerates the decay of C3 and C5 convertases, thus inhibiting C3 activation and deposition on surfaces

Factor D

Circulates in its active form and converts C3 to its active form C3b

Factor H

150

500

Regulates complement activation of the alternative pathway in the fluid phase and on cell surfaces and is a cofactor to factor I. Competes with factor B for binding to C3b. Factor H destroys C3 convertase. It is mainly expressed on the surface of host cells and less commonly on that of microbial cells, making complement activation on the host cells less likely.

Factor I

88

35

Inactivates the classical C3 convertase C4b2a and the alternative convertase C3bBb

Factor P

220

25

Factor P (properdin) stabilizes the alternative convertase C3bBb. Performs a similar function to factor H, since properdin preferentially attaches to microbial rather than host cells.

MCP

45/65

Membrane cofactor protein (MCP, CD46) is a cofactor for the factor I-mediated inactivation of the classical and alternative convertases

SP

80

150

The S protein S (SP) prevents the insertion of C5b-C7 into the cell membrane

AI

The anaphylatoxin inhibitor (AI) inactivates the anaphylatoxins C3a, C4a, C5a

Table 24-2 Reference intervals of complement investigations

Activity /3/

CH50

19.5–60.0 U/mL

C1

1.15 to 4.0 × 1013 eff.mol/mL1)

C2

1.75 to 9.0 × 1011 eff.mol/mL

C3

0.70 to 3.6 × 1013 eff.mol/mL

C4

12.0–60.0 U/mL

CAE

< 60 U/mL

C1-INH

70–130%

Protein concentration (g/l)

C1q

0.05–0.252)

C1-INH

0.15–0.352)

Factor B

0.10–0.402)

C3 /4/3)

C4 /4/3)

Children

3 months

0.67–1.23

0.090–0.305

6 months

0.74–1.38

0.100–0.350

9 months

0.78–1.44

0.115–0.390

12 months

0.80–1.50

0.120–0.400

2–10 years

0.80–1.50

0.125–0.425

12–18 years

0.85–1.60

0.140–0.430

Adults

20 years

0.82–1.60

0.150–0.430

30 years

0.84–1.60

0.160–0.460

40–70 years

0.90–1.70

0.180–0.490

1) eff mol/mL, effective molecules/mL

2) Radial immunodiffusion (Behring, Marburg)

3) Values are 5th and 95th percentiles

Table 24-3 Genetic complement deficiency states /18/

Clinical and laboratory findings

Systemic lupus erythematosus (SLE)

SLE is characterized by polyclonal stimulation of B cells and the occurrence of antibodies of multiple specificities as well as complement activating immune complexes (IC). In healthy individuals, 80% of ICs are bound to CR1 receptors of erythrocytes and B cells and thus circulate in the main bloodstream. This keeps them away from the vessel wall and prevents them from attaching to the latter or to small terminal vessels or vessels of the basal membranes.

In SLE, the elimination of ICs is poor, because:

  • The erythrocytes have become deficient in CR1 receptors. Some patients are also deficient in C4. Due to their poor elimination, the ICs bind to the vessel walls or precipitate on basal membranes. This activates the complement system and exerts an inflammatory destructive effect /19/.
  • Some patients are deficient in C4. C4 is partly responsible for the solubility of ICs and for preventing precipitation /20/.

Laboratory findings: SLE patients with renal involvement almost always have hypocomplementemia, whereas it is less common in extrarenal manifestations of the disease. The classical C3 convertase is activated while CH50, C3 and/or C3c and C4 may be decreased. For disease monitoring, the determination of CH50, C3 and/or C3c is recommended, because a transition from the inactive to the active stage of SLE is more commonly indicated by a decrease in these complement components than by a decrease in C4. Effective therapy with prednisone and cyclophosphamide normalizes CH50, C3 and C4.

Drug-induced lupus erythematosus (LE)

Some therapeutic drugs may induce SLE like symptoms (e.g., hydralazine, isoniazid, and penicillamine). Given in normal doses, they inhibit the covalent binding of C4 to its active binding site. C4a is more strongly affected than C4b. This results in a relative deficiency of C4a with a subsequently reduced elimination of immune complexes. Procainamide, which is metabolized to hydroxylamine, exerts the same effect /19/. The concentrations of C3 and/or C3c or C4 are rarely decreased.

Primary Sjögren’s syndrome (PSS)

PSS is the second most common autoimmune disease after rheumatoid arthritis, having a prevalence of 0.4%. The autoantibodies are mainly directed against the 52 kDa Sjögren’s syndrome antigen A (SSA)/Ro and the 60 kDa Sjögren’s syndrome antigen B (SSB)/La, components of the ribonucleoprotein. Ribonucleoprotein is presented to the immune system during cell apoptosis, viral infections or other pathophysiological events. The antibody response to Ro and La is regulated by the MHC II haplotypes (HLA)DR3 (DRB1*0301) and DR15 (DRB1*1501), which are independent risk factors for PSS. Low levels of C3 and/or C3c or C4 are markers of an unfavorable outcome such as severe disease manifestation, lymphoma, and premature death /21/.

Glomerular renal disease – Generally

Most forms of glomerulonephritis are associated with deposition of immune proteins in the glomeruli. The damage resulting from the formation of immune complexes, in which glomerular or non glomerular antigens are involved, is due to complement activation. By inhibition of complement activation such damage can be reduced or prevented. Conditions with complement activation /22/:

  • Formation of antibodies to an autoantigen. The glomeruli have epitopes which are targeted by autoantibodies. Complement activation is triggered by the resulting immune complexes depositing on the glomerular epithelial cells, leading to the development of membranous nephropathy with proteinuria.
  • Complement can also be activated by circulating immune complexes which cannot be kept in the main bloodstream and get caught in the glomeruli. This mechanism plays a key role in lupus nephritis.
  • Inadequate regulation of complement activation. This is the case in type 1 membranoproliferative glomerulonephritis (MPGN). The autoantibody C3 nephritic factor (C3 Nef) binds to and stabilizes the C3 convertase. This results in the deposition of C3 on surfaces such as the mesangiocapillary structures of the kidneys. During the acute stage, the concentration of C3 is reduced.

– Membranoproliferative glomerulonephritis (MPGN)

Type 1 MPGN: these patients may have a nephritic factor of the terminal complement pathway. It activates the terminal complement pathway by binding to properdin while stabilizing the alternative convertase C3bBbP and creating a binding site for C5. CH50, C3 and/or C3c and C4 are decreased.

Type 2 MPGN: this type is associated with C3 nephritic factor. This factor binds to the initial C3 convertase C3Bb, stabilizes it, and leads to a consumption of C3. As a result of this, formation of cell bound C3 convertase cannot occur, and the terminal complement pathway is not activated.

Type 3 MPGN: may be associated with a decrease in C3 and/or C3c or, less commonly, in C4, but is not thought to be an immune complex disease.

C3 nephritic factor is an IgG autoantibody which binds to and stabilizes the alternative pathway C3 convertase /23/. It promotes the activation of C3 and its deposition on surfaces and leads to the consumption of C3. Type II membranoproliferative glomerulonephritis is the classic disease associated with C3 nephritic factor.

– Post infectious glomerulonephritis (GN)

Endothelial mesangial GN can occur in children days or weeks after infection with β-hemolytic Streptococcus group A, but also Neisseria meningitides and Streptococcus pneumoniae after a latency period of 0.5–3.5 weeks. During the active phase of GN, CH50, C3 and/or C3c and, less commonly, C4 are decreased. C3 hypocomplementemia normalizes after 3–4 months /24/. The prodromes and clinical manifestations in this type of glomerulonephritis are often similar to those in IgA nephropathy (Berger’s disease), but the latter is not associated with hypocomplementemia.

Infectious disease /1/

Inherited complement deficiencies are associated with susceptibility to invasive bacterial infections to a limited spectrum of pathogens, mainly encapsulated bacteria. Late component deficiencies (C5 through C9) are typically associated with recurrent invasive infections caused by N. meningitidis, N. gonorrhoeae and H. Influenzae. Factor D deficiency, which is vary rare, and the properdin deficiency result in selective impairment of alternate pathway function. N. meningitidis is by far most common pathogen encountered in properdin deficiency. Susceptibility to bacterial infection is also important in deficiencies of the classical pathway, but this has gained less attention. In a cohort of C2-deficient persons, invasive infection (meningitis, septicemia) was found in 57% of the patients, and was the predominant clinical manifestation. S. pneumoniae was the most common pathogen /7/.

Rheumatoid arthritis

The determination of C3, C4 and CH50 is of no value for the diagnosis of rheumatoid arthritis, since hypocomplementemia is rarely measured /16/. Decreased levels are most commonly found in juvenile active polyarticular rheumatoid arthritis, where a rise in C4a and Bb correlates with disease activity /27/.

Cryoglobulinemia

Immune complexes that occur in type I, II and III cryoglobulinemia activate the classical C3 convertase; CH50, C3 and/or C3c and C4 are decreased.

Hereditary immunoglobulin deficiency

Approximately 20% of individuals with hereditary IgA deficiency also have homozygous C4a deficiency. A combined deficiency of C4a, an IgG subclass and IgA has also been described /28/.

Hereditary angioedema (HAE) /3031/

HAE has an incidence of 1 : 50.000 and is an autosomal systemic disorder of functional C1 inhibitor protein, a protease inhibitor of the serpin superfamily. The gene C1-INH, which encodes the C1-INH protein, is located on the long arm of chromosome 11, in subregion q12–q13.1. C1-INH is produced in fibroblasts of the liver, in megakaryocytes, monocytes, and in the placenta. There are over 200 known mutants of the gene. Lanadelumab (DX-2930) is a new kallikrein inhibitor with the potential for prophylactic treatment of HAE with C1 inhibitor deficiency /29/.

HAE is classified into two types.

Type I HAE: patients with this type of HAE have normal expression of the C1-INH gene and one abnormal deleted gene. Protein levels and C1-INH activity are 5 to 30%. Type I accounts for 85% of HAE cases.

Type II HAE: patients with this type of HAE have one normal gene and an abnormal gene which codes for dysfunctional C1-INH. The cause are point mutations. Type II accounts for 15% of HAE cases.

Clinical findings: angioedema is the physical manifestation of transient increases in vascular permeability. Bradykinin, generated by activation of the plasma contact system is the mediator of swelling in HAE with C1 inhibitor deficiency. Recurrent episodes of angioedema (subcutaneous or mucosal swelling), gastrointestinal attacks (painful abdominal cramping, circulation related symptoms, vomiting, diarrhea), edemas of the larynx and possibly further organs /30/. HAE differs from urticaria (hives) in that its clinical symptoms usually do not subside within 24 h, but persist for 2–5 days. The lesions are pale and not itchy. Attacks can be triggered by trauma, pressure, emotional stress, menstruation, ovulation, and infectious diseases. Approximately 30% of patients have more than 12 attacks per year, the remainder have fewer attacks. ACE inhibitors lead to recurrent angioedemas in 0.1–2.2% of patients.

C1-INH can be distinguished from type II protein, which is also nonfunctional, by cleavage from albumin using reducing substances before electrophoretic separation /24/. Affected individuals, in most cases women, have normal plasma levels of C1-INH.

Laboratory findings in type I: the C1-INH protein concentration ranges from 0 to 50% of normal and is usually around 20% of the lower reference interval value of 0.15 g/L. During the acute phase of the disease, the serum concentration may be lower, but during the silent phase, it is not a criterion indicating an acute attack. C1-INH activity is usually less than 25% of normal. Due to the deficiency in C1-INH, uncontrolled activation of C1 occurs. Consequently, C4, C2 and CH50 are decreased while the C3 concentration is usually normal and less frequently decreased.

Laboratory findings in type II: the C1-INH protein concentration is normal or even elevated. Functional determination of C1-INH usually shows a marked decrease in activity, e.g. down to 0.09 C1-INH/mL (reference interval 0.8–1.25). CH50, C2 and C4 are also decreased. A type III is differentiated from type II HAE. In type III, nonfunctional C1-INH protein is bound to albumin. Type III, nonfunctional C1-INH protein can be differentiated electrophoretically from nonfunctional type II C1-INH protein and is detached from albumin by reducing reagents /24/. The affected, mostly women, have a normal plasma concentration of C1-INH.

Table 24-4 Hereditary deficiency of complement components /24/

Clinical and laboratory findings

Deficiency of C1q, C1r, C1s

Deficiency does not lead to increased susceptibility to infection. However, affected individuals have a high incidence of autoimmune diseases, in particular lupus erythematosus.

Deficiency of C2

C2 deficiency is the most common complement deficiency. While this deficiency does not normally cause problems, affected individuals have a higher than normal incidence of infections, in particular during periods of increased exertion or stress. They also have an increased incidence of autoimmune diseases.

Deficiency of C3

C3 deficiency is a major defect because it is the point where the three complement pathways intersect and C3 is the major opsonin of the complement. C3 deficient individuals often have frequent infections with high grade pathogens like S. pneumoniae, Neisseria meningitides and Enterobacteriaceae. They often also have autoimmune diseases, in particular glomerulonephritis. Approximately 50% of patients with hereditary C3 deficiency or a deficiency of components of the classical activation pathway (C1, C2 and C4) present with SLE or an SLE like disease /25/.

Deficiency of C4 /20/

C4 exists in two major forms or isotypes (C4a and C4b). They are encoded by the C4A and C4B genes within the MHC complex which differ in their biochemical and functional characteristics. Both proteins participate in the solubilization of immune complexes (ICs) and the prevention of immune precipitation. The thioether bonds of C4a preferentially transacylate onto amino groups on ICs, while C4b binds more avidly to hydroxyl groups on carbohydrate bearing surfaces. Hereditary C4 deficiency (C4*QO) predisposes to SLE.

  • Heterozygous deficiency of C4a, due to a large deletion of the C4A and 21-hydroxylase A (21-OHA) genes on the Caucasian HLA-B8, DR3 haplotype, occurs in over 50% of white patients with lupus.
  • Homozygous deficiency C4A is found in 13–15% of whites with SLE and 2% of controls. Partial or complete deficiency of C4B may also contribute a risk factor.
  • Total C4 deficiency due to homozygosity for both null alleles at both C4A and C4B, albeit rare, is associated with SLE in over 80% of instances.

Deficiency of C5–C9

Individuals with C5, C6, C7, C8 or C9 deficiency have a higher prevalence of autoimmune diseases and infections with Neisseria than the general population. Infectious episodes or sepsis generally only occur from mid childhood onwards in these patients. C6 deficiency is more common in blacks than in whites. C9 deficiency is common in the Japanese population.

Deficiency of properdin

Properdin is encoded by a gene on the X chromosome. In type 1 properdin deficiency, the most common inherited type, the individuals are unable to secrete the protein. Type 2 is due to a point mutation in which the properdin secreted has no functional activity, while in type 3 it has reduced functional activity. Individuals who are deficient in properdin have an increased incidence of infection with Neisseria meningitides and of autoimmune diseases.

Deficiency of factor H

This is the control protein for C3. Factor H deficiency has been reported to be associated with hemolytic-uremic syndrome.

Deficiency of complement receptors

CR1 (CD35): this is the receptor for C3b and C3bi on cells, promotes phagocytosis and regulates C3 degradation. Patients with SLE are deficient in erythrocyte CR1. It is believed that this is a genetic defect which predisposes to immune complex disease. All patients with immune complex disease have an acquired deficiency of erythrocyte CR1; the receptors were likely removed when the immune complexes were cleared from the erythrocytes by macrophages /25/.

CR2 (CD21): the antigen-antibody reaction triggers the activation of complement, and C3 cleavage products are deposited on the surface of microorganisms. Some of these fragments, such as C3d, are recognized by the CR2 receptors on B cells. The simultaneous presence of antigen and C3d on the surface of bacteria allows dual molecular antigen recognition by B cells via the antigen receptor and CR2. The CR2-mediated signal is important for B cell activation. Decreased expression of CR2 on B cells is found in patients with SLE. An acquired decrease in CR2 expression is seen in HIV-infected individuals /26/.

CR3: CR3 is a heterodimer consisting of CD11b and CD18. Endothelial CD18 receptors are up regulated at infection sites. Defective or absent CD18 prevents the migration of polymorphonuclear granulocytes (PMNs) to the site of infection, which results in local pyogenic infections. Paradoxically, leukocytes are elevated since the PMNs are unable to migrate out of the vessels /27/.

Deficiency of complement regulatory proteins CD55, CD46, CD59

CD55, CD46 and CD59 are glycosylphosphatidylinositol (GPI) anchored membrane proteins. CD55 and CD59 prevent hemolysis because erythrocytes are protected from being attacked by the complement system. Relevant complement regulatory proteins are /3438/. Decay accelerating factor (DAF, CD55) that accelerates the decay of cell surface bound C3 convertases, thus limiting the formation of the C5 convertase and formation of the membrane attack complex

  • Membrane cofactor protein (MCP, CD46) that accelerates decay of C3 convertases. In conjugation with soluble factor I, CD46 also inactivates C3b to iC3b, thereby preventing reformation of C3 convertase; erythrocytes do not express CD46
  • CD59 prevents C8 from interacting with C9 on the erythrocyte membrane, thereby blocking the formation of membrane attack complex: CD59 deficiency is always associated with significant hemolysis.

The GPI anchor is encoded by a gene on the X chromosome known as phosphatidylinositol glycan A (PIGA). The glycolipid moiety anchors more than a dozen proteins to the cell surface of blood cells /34/.

Refer to Fig. 24-6 – Regulation of complement pathway by control proteins.

Deficiency of mannan binding lectin (MBL) polymorphism

Up to 5% of the population have a certain degree of MBL deficiency. Affected individuals are generally healthy, but in children low levels can be associated with an increased risk of bacterial infections, in particular at the age of 6 months, when passive maternal protection is lost and the infant’s own immune system is not yet able to produce sufficient immunoglobulin. Bone marrow transplanted patients who carry the MBL polymorphism also are at increased risk of bacterial infections /27/.

Table 24-5 Acquired complement deficiency

Clinical and laboratory findings

Immune complex disease /2/

Complement promotes the clearance of foreign antigens through immune complex formation. C1q, C4b, and C3b coat the immune complex, maintain its solubility and provide ligands for the complex to attach to cells. There are several conditions leading to depletion of complement in serum. Depletion includes C3 and C4 binding autoantibodies. Another factor is the binding of C3b to complement receptor 1 on erythrocytes, that than carry the complex to the liver and spleen where they are eliminated by the resident macrophages. Such and other pathomechanisms result in the reduction of complement in serum and a decrease of CH50. Beyond these examples see Tab. 24-6 – Acquired complement deficiency characterized by immune complexes.

Autoantibody syndrome /2/

In autoantibody syndrome the autoantibody binds to a fixed antigen on cell or tissue targets and the combination of both immune reactants damage the target. This is the case in patients with e.g., acquired angioedema, C3 nephritic factor or in all patients with HUVS.

Acquired angioedema (AAE) /3032/

Patients with AAE generally have normal C1-INH synthesis, but catabolize the protein faster than normal. Usually there is no family history, and patients with AAE are older than those with HAE at initial presentation. Some patients have autoantibodies or B cell disease with or without synthesis of monoclonal antibodies, or a malignant lymphoma. These patients often present because of an angioedema. The malignant plasma cells or lymphoma cells have bound C1q on their surface, leading to complement activation and consumption of C1-INH. Another important pathophysiologic mechanism in the development of AAE is the fact that anti-C1-INH antibodies interact with the complex of C1-INH and its bound protease (C1q or C1r), leading to the degradation of the 110 kDa C1-INH molecule into a 96–98 kDa protein. This results in increased activation of the complement system and the kallikrein-kinin system with increased production of bradykinin.

Laboratory findings: AAE is characterized by deficiency of C1, C4, C3 and normal or reduced concentration of nonfunctional C1-INH.

Membranoproliferative glomerulonephritis

C3 nephritic factor is an autoantibody against the alternative pathway convertase and results in secondary C3 deficiency. Children are most often affected and present with the triad of membranoproliferative glomerulonephritis, partial lipodystrophy, and frequent bacterial infections /33/.

Paroxysmal nocturnal hemoglobinuria (PNH) /34/

PNH cells are susceptible to hemolysis because of a loss of 2 complement regulators, decay accelerating factor (CD55) and CD59, the inhibitor of membrane attack complex (MAC). Both of these regulators are tethered to the cell membrane by a glycosylphosphatidyl and inositol anchor (GPI). In nearly all cases, GPI anchor deficiency results from a mutation in the gene PICA whose product is required for the first step in GPI anchor synthesis. This results in the absence of complement regulatory proteins CD55 and CD59, leading to chronic complement-mediated hemolysis of the PNH erythrocytes. Hemolysis in PNH is chronic because of a continuous state of complement activation through tick-over, but paroxysms resulting in brisk hemolysis coincide with increases in complement activation that may occur with inflammatory states or surgery. For further information Refer to Tab. 15.3-11 – Classification and differentiation of normocytic anemia.

Atypical hemolytic uremic syndrome (aHUS) /34/

AHUS is a thrombotic micro angiopathy that presents with intravascular hemolysis, thrombocytopenia and acute renal failure. Most cases of aHUS are caused by mutations in genes encoding proteins involved in the function of activated protein C (APC) or by autoantibodies directed against APC regulatory proteins (Fig. 16.1-7 – Plasma coagulation system with positive and negative feedback mechanisms). These mutations can effect proteins that help degrade cell surface C3b (APC inhibitors), such as factor H (most common), factor I, monocyte chemoattractant protein (MCP), and thrombomodulin or proteins that drive the APC (activating mutations) in C3 or factor B. The main cause of dysregulation of the alternative complement pathway are mutations in factor B (D254G and K325N), the main regulator of C3 convertase. The mutant proteins form a high affinity binding site for C3, leading to a hyper functional C3 convertase which is resistant to decay by factor H (Fig. 24-6 – Regulation of the complement pathways by control proteins). This leads to increased deposition of C3 on glomerular cells and formation of membrane attack complex. In about 50% of aHUS patients the familial form of the disease exists (penetrance is about 50%) other cases have a putative trigger (e.g., pregnancy, infection, surgery) that leads to infection and presumably increases complement activation.

Capillary leak syndrome

Patients with a non genetic deficiency in C1-INH can display C1-INH deficiency during the course of a generalized inflammatory response. Such a deficiency state is thought to be responsible for the capillary leak syndrome /17/. This syndrome may occur in conjunction with septic shock, interleukin-2 therapy, severe burns, and after bone marrow transplantation. Clinical manifestations may include generalized edema, ascites, pre renal kidney failure, and hypovolemic shock that is refractory to fluid resuscitation. The clinical condition can be improved by administering C1-INH concentrate.

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.

Table 24-6 Acquired (secondary) complement deficiency characterized by immune complexes /18/

Clinical and laboratory findings

Embolism

Spontaneously occurring embolism or atheromatous embolism induced by introduction of a catheter results in activation mainly of the alternative C3 convertase. Hypocomplementemia occurs only briefly during and after the event. Affected patients may present with manifold symptoms such as vasculitis like, transient skin rashes, renal failure, gastrointestinal bleeding, eosinophilia, and thrombocytopenia.

Sepsis

While systemic infectious diseases cause elevated complement levels, sepsis syndrome results in activation of the classical C3 convertase. The activation is due to proteolysis of C1-esterase inhibitor. There is increased production of C5a, which leads to neutrophil paralysis /13/.

Acute pancreatitis

Pancreatic proteases convert inactive complement components into their active forms and thus activate the C3 convertases. CH50, C3 and/or C3c and C4 are decreased. Within a few days, concentrations normalize again.

Liver failure

In fulminant hepatitis there is decreased production of C3 and C4, but C1q levels are normal.

Malnutrition

Severe malnutrition (involving both a deficient nutrient intake as well as an inadequate food composition), especially in children, leads to a decrease in C3 and C1q whereas C4 is normal.

Nephrotic syndrome

C3 and/or C3c and C4 are normal, whereas factor B, C1q, C2, C8 and C9 may be decreased.

Myocardial infarction

Slight decreases in C3 and/or C3c and C4 have been described.

Burns

Burns affecting more than 25% of the body’s surface area lead to reduced complement levels.

Hemolytic uremic syndrome (HUS), Thrombotic thrombocytopenic purpura (TTP)

HUS and TTP are syndromes of micro angiopathic hemolytic anemia and thrombocytopenia, which are both associated with occlusion of the microcirculation of organs. In children with renal insufficiency the term HUS is used, while in adults with predominantly neurological symptoms the condition is referred to as TTP.

Although the two disorders differ in their clinical manifestations, histologically they present with the same vascular lesion (i.e., widening of the subendothelial space and intravascular thrombocyte aggregates). The consumption of thrombocytes and destruction of erythrocytes lead to thrombocytopenia and anemia. Approximately 80–90% of patients have acute symptoms which regress spontaneously. The conditions can be caused by verotoxins produced by E. coli, as well as by medications or other diseases /35/.

Laboratory findings: approximately 73% of patients with HUS or TTP have a decreased C3 concentration. This is also the case in 23% of their relatives. These relatives with decreased C3 levels have a 17-fold higher risk of developing HUS or TTP than relatives with normal C3 levels, and a 28-fold higher risk than the general population /35/.

Cardiopulmonary bypass

Slight decrease in C3 and/or C3c and C4 due to C3 convertase activation induced by the nylon grid of the oxygenator and the oxygenation of blood.

In vivo diagnostic agents

X-ray contrast media may cause a slight decrease in C3 levels. Angiographic reagents but also non ionic X-ray contrast media may activate the alternative C3 convertase. Heparin-protamine complexes activate the classical C3 convertase while inulin activates the alternative C3 convertase.

Malaria

In malaria, the causes of a decrease in C3 and/or C3c and C4 during a hemolytic episode are multifactorial.

Herpes simplex infection

Due to the activation of alternative C3 convertase, C3 and/or C3c may be reduced whereas C4 is normal.

Porphyria

Activation of alternative C3 convertase in patients with erythropoietic protoporphyria results in a decrease in C3 and/or C3c levels after sun exposure.

Graves’ disease, thyroiditis

The occurrence of thyroglobulin containing immune complexes, immune complex disease, and decreases in C3 and/or C3c, C4 and CH50 have been described.

Liver disease

In liver disease, complement activation is common but is rarely detectable based on C3 or C4 concentration or measurement of CH50. In decompensated liver cirrhosis, C3, C4, CH50 and AP50 are decreased. Such a state is associated with increased risk of infections as well as increased mortality /36/.

Ileo-jejunal bypass

Immune complex mediated arthritis, hemolysis, and decreases in C3 and/or C3c and C4 are possible.

Chemotherapy of malignant tumor

During the treatment of Hodgkin’s disease and lymphoblastic leukemia with chemotherapy, immune complexes and decreased levels of C3 and/or C3c and C4 may occur.

AIDS

HIV activates the classical complement pathway by C1q binding to the GP41 transmembrane protein of infected cells. In AIDS patients, the hypocomplementemia (as confirmed by C3, C4 concentration or CH50 measurements) may be due to HIV induced complement activation or caused by opportunistic infections /37/.

Multiple myeloma

Malignant degeneration of plasma cells with synthesis of monoclonal antibodies can lead to the production of anti idiotypic antibodies which bind to the monoclonal antibodies to form immune complexes. If C1 is activated, increased consumption of C1-INH, C4, and C2 occurs. C3 and/or C3c are not decreased, because production of C3 convertase is impaired.

Glomerulonephritis in chronic infections

Chronic bacterial infections such as endocarditis, soft tissue, pulmonary or hepatic abscesses as well as viral infections such as hepatitis C may be associated with circulating immune complexes, decreased C3 and C4 levels, GN, and vasculitis. Chronic infections with spirochetes and parasites are also thought to cause these symptoms. Severe forms of GN, vasculitis, and complement decrease are only seen in hepatitis B.

Table 24-7 Determination of CH50 and AP50 and interpretation (examples) /23/

Complement
pathway

AP50

CH50

Deficiency

Alternative
defective

R

N

Properdin, Factor B, H

Classical
defective

N

R

C2

Terminal lytic
complex defective

R

R

C6

Sampling error

R

R

Artifact

R, reduced; N, normal

Table 24-8 Determination of C3 and C4 and interpretation (examples) /23/

Complement
pathway

C3

C4

Disease

Classical
defective

N

R

R

R

SLE, cryoglobulins

C1-INH deficiency

SLE and nephritis

HUVS

Alternative
defective

R

N

Bacterial infection

Nephritic factor

Factor H deficiency

SLE, systemic lupus erythematosus; HUVS, hypocomplementemic urticarial vasculitis syndrome; N, normal; R, reduced

Table 24-9 Activation pathway in the presence of hypocomplementemia /2439/

Disease

Activation pathway

alternative

classical

Bacterial sepsis

  • Gram negative

3

1

  • Pneumococci

2

2

Viral disease

  • Dengue fever

 

3

  • Hepatitis B

 

2

Disseminated
cryptococcosis

2

 

Malaria

1

2

Trypanosomiasis

2

2

Rheumatic diseases

  • Active SLE

1

3

  • Rheumatoid arthritis*

 

2

  • Serum sickness

 

1

  • Vasculitis

 

2

Hematologic diseases

  • Transfusion reaction

 

3

  • Dialysis related neutropenia

2

 

Renal diseases

  • MPGN (with C3 Nef)**

3

  • Post streptococcal nephritis

2

Miscellaneous

  • Hereditary angiodema

 

4

  • C3 inactivator deficiency

4

 

  • Urticaria

 

1

1, described; 2, occasionally; 3, often; 4, usually; * with joint effusion; ** membranoproliferative glomerulonephritis

Table 24-10 Regulation of the complement system by control proteins

Regulation

C1 activation

The activation of this step is controlled by C1-esterase inhibitor (C1-INH). This protein inhibits the proteolytic activity of C1r for the cleavage of C1s. The cleavage of C4 and C2 can also be inhibited.

Convertases, classical C4b2a, alternative C3bBb

Regulation of the convertases is accomplished by 5 control proteins, 3 of which are co factors of factor I, which is capable of inactivating both convertases. The following control proteins act on the convertases:

  • C4bp: C4b-binding protein acts a cofactor for factor I and inactivates the C4b2a convertase by binding to C4b and by accelerating the dissociation of C2a. This reduces half life (normal 3 min.). The resulting C4b-C4bp complex is the substrate of factor I.
  • Factor H: acts as a cofactor for factor I and binds to the C3bBb convertase while releasing the C3b-H complex, which is the substrate of factor I. The normal 90 sec. half-life of the convertase is significantly shortened.
  • CR1: is the complement receptor of blood cells and is also considered a cofactor of factor I. C3b is bound by the complement receptors and cleaved by factor I to yield inactive fragments such as C3c and C3dg.
  • DAF (decay accelerating factor): protein of the tissue cell membrane. It prevents the formation of a cell bound C3 convertase, promotes its decay, and thus protects cells from complement attack.

C5b-C9 membrane attack complex

The following control proteins are known:

  • S protein (vitronectin): when in solution, S-protein binds to C5b-C7, thereby preventing the complex from penetrating the cell membrane
  • HRF (homologous restriction factor): prevents formation of the transmembranous channel
  • 18-kDa protein (CD59): cell membrane protein which modulates membrane insertion of the membrane attack complex in such a way that erythrocytes coated with C5b-C7 cannot be hemolyzed.

Table 24-11 Control proteins on the cell membrane of blood cells

Function

CR1

Receptor on erythrocytes, granulocytes and monocytes; CR1 is a cofactor of factor I and binds C3bBb, allowing the latter to be inactivated by factor I.

CR2

Receptor of B cells, binds C3b and causes a proliferation of B cells.

CR3

Receptor on granulocytes and monocytes, binds C3bi, activates these cells to phagocytize C3bi-coated particles. CR3 is identical to the adhesion protein Mac-1.

DAF (decay accelerating factor)

Prevents assembly of alternative C3bBb convertase on the cell surface and accelerates decay of the enzyme.

HRF (homologous restriction factor)

Prevents transmembrane channel formation by the membrane attack complex.

CD18

Modulates membrane insertion of the membrane attack complex.

Figure 24-1 Pathways activating the complement system, modified according to Ref. /22/. Abbreviations and further information refer to Tab. 24-1 – Proteins of the complement system.

Classical pathwayAg:Ak Lectin pathwayCarbohydrates Alternative pathwayBacteria,damaged cells DAF,CR1 (C3 convertase)C4b2a (C5 convertase)C4b2a3bC3bBb3bP C3a +C3 +C3 C3b C5 C5a C5b-8 CD59 +(C9)n C5b-9 C5b + C6,C7,C8 Enhancer loop MCPCR1 C3bBbP

Figure 24-2 The role of C1-INH in the regulation of fibrinolysis, the classical pathway of the complement system, and the bradykinin system. With kind permission from Ref. /39/.

Factor XII F XIIa, F XIIf C1-INH C1-INH Fibrinolysis Plasminogenproactivator Plasminogen C1-INH C1-INH ConversionActivationInhibition Plasmin Plasminogenactivator C1-INH C1-INH C1-INH C1-INH Complement system Bradykinin HMWK C1-INH Kallikrein Prekallikrein C1 C1 C4 C2 C3 C4 C5b C9 C4a C3a C5a C1-INH C1-INH Contact system

Figure 24-3 Classical pathway, lectin pathway and alternative pathway of the complement system. In the classical pathway, the complement protein C1q is activated by its binding to immune complexes. The lectin pathway is activated by the binding of carbohydrate molecules of the bacterial cell wall to mannose binding protein (MBP). This activates MBP associated serine protease (MASP). The complex bound MASP is able to cleave C4 and C2 and as a consequence, C3 is cleaved by C4b2a convertase. The alternative pathway is activated by surfaces such as bacterial cell walls.

Lectinpathway Carbohydrates Classicalpathway Alternativepathway Native C3b Immune complexes Activated C1 Surfaces-boundedC3b Activatingsurfaces(Bacteria) C4b2a(C3 convertase) C4b2a(C3 convertase) C3bBbP(C3 convertase) Feedback Factors B,DProperdin (P) Membraneattack complex C6, C7, C8, C9 C5b-9Lysis of cellmembrane C1 (C1q, r, s) C4, C2 C4, C2 C3 MASP MBP C3a C5a C3b C3b C5b

Figure 24-4 Activation of the classical complement pathway and formation of the membrane attack complex, modified from Ref. /15/.

C1 s C4 C4 a C2 C2 b C5 C3 convertase C4b2a C3 C3 b C3 a C5 a C5 b C5 convertase C6, C7 C8, C9 C5b-9 complex C1 Immunocomplex IgM, IgG 1–3 +

Figure 24-5 Activation of the alternative complement pathway and formation of the membrane attack complex /16/.

Ba C3 enhancer convertase C3 (H 2 O) Bb C3 C3 a C3 b Activating surface Amplification C3bB B, Mg 2+ Cell-bound C3 convertase C3bBb (P) C3 b C5 C5 convertase Ba C5 a C5 b C5b-9 complex C3 (H 2 O) + B Mg ++ C3 (H 2 O) B D D, P

Figure 24-6 Regulation of the classical complement pathway (top) and the alternative complement pathway (bottom) by control proteins. The control proteins are shown in frames. AI, anaphylatoxin inactivator. Figure modified according to Ref. /16/.

C1-INH DAF C4b2a C4bp CR 1 C3bBb H DAF C3 C3b C3 a C5 a C5 b C5 AI C5b-9 complex S proteinHRF18 kDa protein I C4 C2 C3, B, D C1
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