Complement Regulatory Proteins and Anaphylatoxins

System Controls

• Complement activation can cause tissue damage and systemic effects if uncontrolled.
• Plasma proteins act as system regulators to ensure infectious agents and not self-antigens are destroyed, localizing the reaction.
• Specific cell receptors also control the activation process.
• Approximately one-half of complement components control critical activation steps.
• Majority of control proteins target halting accumulation of C3b because C3 activation is the key step in all pathways.
• Controls exist at all crucial steps.

Plasma Complement Regulators (Table 7-2)

• C1 inhibitor (C1-INH): Molecular weight: 105 kD, Concentration: 240 mg/mL, Function: Dissociates C1r and C1s from C1q.
• Factor I: Molecular weight: 88 kD, Concentration: 35 mg/mL, Function: Cleaves C3b and C4b.
• Factor H: Molecular weight: 150 kD, Concentration: 300-450 mg/mL, Function: Cofactor with I to inactivate C3b; prevents binding of B to C3b
• C4-binding protein (C4BP): Molecular weight: 520 kD, Concentration: 250 mg/mL, Function: Acts as a cofactor with I to inactivate C4b.
• S protein (vitronectin): Molecular weight: 84 kD, Concentration: 500 mg/mL, Function: Prevents attachment of the C5b67 complex to cell membranes.

Regulation of Classical and Lectin Pathways

• C1 inhibitor (C1-INH) inhibits activation at the first stages of both classical and lectin pathways.
• Primary role of C1-INH is to inactivate C1 by binding to the active sites of C1r and C1s.
• C1r and C1s instantly and irreversibly dissociate from C1q.
• C1q remains bound to antibody, but all enzymatic activity ceases.
• C1-INH also inactivates MASP-2 binding to the MBL-MASP complex, halting the lectin pathway.
• C1-INH: Glycoprotein w/ molecular weight of 105,000, mainly synthesized in the liver; monocytes may also be involved in its manufacture.
• Further formation of C3 convertase in classical and lectin pathways is inhibited by four main regulators.
  • Soluble C4-binding protein (C4BP).
  • Cell-bound receptors: complement receptor type 1 (CR1), membrane cofactor protein (MCP), decay-accelerating factor (DAF).
• All act with Factor I, a serine protease that inactivates C3b and C4b when bound to one of these regulators.
• C4BP occurs abundantly in plasma and has a molecular weight of about 520,000.
• C4BP: Capable of combining with either fluid phase or bound C4b; C4b cannot bind to C2 & is made available for degradation by Factor I.
• If C4BP attaches to cell-bound C4b, it can dissociate it from C4b2a complexes, causing the cessation of the classical pathway.
• CR1, also known as CD35: Large polymorphic glycoprotein with molecular weight between 165,000 and 280,000.
• CR1 mainly found on peripheral blood cells (neutrophils, monocytes, macrophages, erythrocytes, eosinophils, B & some T lymphocytes, and follicular dendritic cells).
• CR1 binds C3b and C4b by affinity, binding C3b greatest.
• Once bound to CR1, both C4b and C3b can be degraded by Factor I.
• CR1's main function is as a receptor on platelets and red blood cells (RBCs), mediating transport of C3b coated immune complexes to the liver and spleen.
• Fixed tissue macrophages strip immune complexes from the RBCs, process complexes, and return the RBCs intact to circulation.
• The ability of cells to bind complement coated particles is called immune adherence.
• MCP, or CD46: Molecular weight between 50,000 and 70,000, found on practically all epithelial and endothelial cells except erythrocytes.
• MCP: the most efficient Factor I-mediated cleavage cofactor of C3b, can serve as a cofactor for cleavage of C4b, but is not as effective as C4BP.
• MCP also helps to control the alternative pathway because binding of Factor B to C3b is inhibited.
• DAF, or CD55: a 70,000 d membrane glycoprotein, third main receptor with wide tissue distribution.
• DAF found on peripheral blood cells, endothelial cells and fibroblasts, and numerous types of epithelial cells.
• DAF can dissociate both classical and alternative pathway C3 convertases and bind both C3b and C4b similarly to CR1.
• DAF does not prevent initial binding of C2 or Factor B to the cell but can rapidly dissociate both from their binding sites, blocking the assembly of an active C3 convertase.
• Carboxy-terminal portion of DAF is covalently attached to a glycophospholipid anchor inserted into the membrane lipid bilayer's outer layer.
• DAF mobility in the membrane allows it to reach C3 convertase sites not immediately adjacent to it.
• Having DAF on host cells protects them from bystander lysis.
• Discrimination of self from nonself is a main mechanism; foreign cells do not possess this substance.
• C3b or C4b is not permanently modified, but they can re-form elsewhere as active convertases.

Regulation of the Alternative Pathway

• The main soluble regulator of the alternative pathway is Factor H, having a molecular weight of 160,000.
• Factor H binds to C3b, preventing Factor B binding.
• C3b: Fluid phase has hundredfold greater affinity for Factor H than Factor B, but on cell surfaces C3b preferentially binds to Factor B.
• Factor H accelerates the dissociation of the C3bBb complex on cell surfaces.
• When Factor H binds to C3bBb, Bb becomes displaced.
• C3 convertase activity is reduced in plasma and on cell surfaces with Factor H.
• Factor H acts as a cofactor allowing Factor I to break down C3b.
• Apparently, only those molecules with tightly bound Factor H acquire high-affinity binding sites for Factor I.
• When Factor I binds, a conformational change takes place that allows it to cleave C3b.
• On cell surfaces, C3b is cleaved into C3f, released into the plasma, and iC3b, which remains attached but inactive.
• iC3b is further broken down to C3c and C3dg by Factor I with another cofactor: the CR1 receptor.
• Key complement regulation role, Factor H has recently been shown to play a role in a variety of disorders.

Regulation of Terminal Components

• S protein: soluble control protein acting at a deeper level of complement activation.
  • Otherwise known as vitronectin, the molecule interacts with the C5b-7 complex as it forms in the fluid phase, preventing it from binding to cell membranes.
  • Binding of C8 and C9 still proceeds, but polymerization of C9 does not occur, meaning the complex is unable to insert to itself into the cell membrane or to produce lysis.
• A receptor blocks formation of the MAC, known as various terms, including membrane inhibitor of reactive lysis (MIRL) or CD59.
• MIRL is widely distributed on cell membranes of all circulating blood cells, including RBCs, and many other endothelial, epithelial types of cells.

Complement Receptors and Their Biological Roles

• Complement receptors found on host cells amplify and enhance the immune response by augmenting phagocytosis and stimulating accessory cells.
• CR2/CD21: found on B lymphocytes, follicular dendritic cells.
• CR2/CD21: Ligands include C3b degradation products (C3dg, C3d, iC3b).
• Epstein-Barr virus enters B cells by binding to this receptor.
• CR2 is present only on mature B cells and is lost when they convert into plasma cells.
• CR2 is important as the B-cell co-receptor for antigen; with CD19, it binds complement-coated antigen and cross-links to membrane immunoglobulin to activate B cells.
• Immune complexes enhance B-cell differentiation and production of memory cells more effectively than antigen alone.
• CR3 (CD11b/CD18): Found on monocytes, macrophages, neutrophils, and natural killer (NK) cells, binding particles opsonized with iC3b.
• CR3 binding action: calcium-dependent.
• CR3 receptor mediates phagocytosis of particles coated with complement fragments.
• CR3 proteins can trigger surface adhesion and increased phagocytic activity in phagocytic cells.
• Patients lacking these receptors show impairment of chemotaxis, surface adherence, and aggregation.
• Deficiencies in phagocytosis are also noted since these individuals have impaired capacity to bind iC3b-coated particles: subject to recurrent infections.
• CR4 (CD11c/CD18): Very similar that it also binds iC3b fragments in a calcium-dependent fashion.
• CR4 is found on neutrophils, monocytes, tissue macrophages, activated T cells, NK cells, dendritic cells, activated B cells.
• Function of CR4 appears similar to that of CR3 since they may assist neutrophil adhesion to the endothelium during inflammation.
• Receptors specific for C1q are found on neutrophils, monocytes, macrophages, B cells, platelets, & endothelial cells.
• These receptors, known as collectin receptors, bind the collagen portion of C1q and enhance C1q binding to FC receptors.
• Receptors interacting only with bound C1q seem to increase phagocytic cells' uptake of immune complexes opsonized with C1q.
• Receptors may also act on neutrophils to enhance the respiratory burst triggered by IgG binding to FC receptors.

Biological Manifestations of Complement Activation

• Complement activation is a very effective means of amplifying the inflammatory response to destroy and clear foreign antigens.
• The cycle does not always have to proceed to lysis; initiators can be more abundant than MAC formers.
• Complement proteins also link innate and adaptive immunity.
• They act as opsonins to facilitate recognition and destruction by phagocytic cells, and they facilitate B-cell activation to uptake and present antigens.
• Molecules can be anaphylatoxins, chemotaxins, and opsonins.
• Anaphylatoxin: small peptide that increases vascular permeability, contracts smooth muscle, and releases histamine.
• Proteins that play part are C3a and C5a that have molecular weights between 9000 and 11,000 and are formed as cleavage products from larger complement components.
• C5a: most potent molecule, >200 times more powerful than C3a.
• C3a and C5a attach to receptors on neutrophils, basophils, mast cells, eosinophils, smooth muscle cells, and vascular endothelium.
• C3a attaches to receptor (C3aR) & C5a receptor (C5aR).
• When binding occurs on basophils and mast cells, histamine is released, that increases vascular permeability and causing contraction of smooth muscles.
• C5a causes neutrophils to release hydrolytic enzymes, oxygen radicals, and prostaglandins, aiding in destruction of foreign antigens.
• C5a also serves as a chemotaxin for neutrophils, basophils, eosinophils, mast cells, monocytes, & dendritic cells (directed to source of antigen concentration).
• Vascular permeability increased so neutrophils migrate from blood vessels to the tissues & tend to aggregate.
• Binding of C5a to monocytes causes oxidative burst that increased production of hydrolytic enzymes, neutrophil chemotactic factor, platelet activating factors, interleukin-1 (IL-1), & toxic oxygen metabolites.
• Activation causes fever and an increase in acute-phase reactants, both characterize an inflammatory response.
• C3a and C5a are rapidly inactivated by carboxypeptidase N in the plasma to control and localize effects.
• C3a is cleaved in seconds, but conversion of C5a occurs more slowly.
• Last major effect of complement-derived peptides is opsonization.
• C4b, C3b, iC3b, and C3dg accumulate on cell membranes as complement activation and proceeds, bind to specific receptors on erythrocytes, neutrophils, monocytes, and macrophages.
• Binding facilitates phagocytosis and clearance of foreign substances/debris, a key functions of the complement system.
• C3 product attachment to an antigen enhances the B-cell response.

Complement and Disease States

• Complement acts as a powerful weapon to combat infection by amplifying phagocytosis but can contribute to tissue damage or death.
• Harmful if: systemically activates greatly such as in gram-negative septicemia, activated by tissue necrosis due to myocardial infarction, causes lysis of red blood cells.
• Septicemia caused by gram-negative organisms generates large quantities of C3a and C5a that lead to neutrophil aggregation and clotting.
• The tiny pulmonary capillaries and interstitial pulmonary edema may suffer damage.
• Tissue injury following blood supply obstruction, like in a myocardial infarction or heart attack, causes complement activation and deposition of MACs on cell surfaces.
• Receptors for C3a and C5a have been found in coronary plaques, indicating that complement components may increase damage to heart tissue.
• Other lysis may be another end-result of complement activation.
• Hemolytic diseases like cold autoimmune hemolytic anemia are characterized by antibody presence the binds at low temperatures.
• Activation causes a need to warm up, complement fixation, and ultimate lysis.

Major Pathway Components

• Excess activation of the complement system can result in disease states that also lack individual components; causes deleterious effect.
• Except for C9, hereditary deficiency of any complement manifests in increased susceptibility to infection and delayed immune complex clearance.
• Most conditions are inherited on autosomal recessive gene, making them rare (approx. 0.03% of the general population).
• Lack of C2 (most common deficiency) found in 1 in 20,000 individuals.
• Recent evident states atherosclerosis is related to a C2 deficiency.
• C2-deficient individuals may be more prone to recurrent streptococcal and staphylococcal infections.
• Because Factor B locus is nearby, C2-deficient persons are often reported with Factor B decrease.
• MBL the next to follow the occurring deficiency frequency.
• Lack of MBL is associated with pneumonia, sepsis, meningococcal disease in infants.
• Low MBL is also assocaited with some cancers/infections during chemotherapy/autoimmune disorders like systemic lupus erythematosus (SLE) with connections yet to be defined.
• Deficiency of C3: Important mediator for all common pathways however, C3 deficiencies are very rare.
• Having this lack, a developer is prone to severe & recurrent, life threatening infections via encapsulated bacteria like Streptococcus pneumoniae & may is subject it self in other complex immune diseases.
• Complexes here can result lodge in kidneys and may result in glomerulonephritis.
• Any terminal component deficiency of cascade C5 – C8 is shown to increased susceptibility for systemic Neisseria infections; this is meningococcal meningitis and disseminated gonorrheal disease.

Regulatory Factor Components

• Paroxysmal nocturnal hemoglobinuria (PNH) is caused by missing/defective regulatory component;Individuals have RBCs deficient in DAF.
• RBCs: Subject to lysis by bystander effect once triggered
• Individuals: Shown defiencey- glycophospholipid anchor of DAF molecule, as prevents being inserted into cell membrane.
• C3b deposition is activated on erythrocytes through activation of either pathway - Result -> complement-mediate intra/extra vascular hemolysis and/or to the resulting of having chronic hemolytic anemia.
• Deficiency of DAF associated with lack of CD59 (MIRL) and both are implicated in PNH.
• CD59 has the same glycophospholipid anchor found in DAF; the gene deficiency affects both molecules.
• CD59: Prevents to inserting C9 into membrane via binds C5b-8 comple that will inhibit -> transmembrane channels formation.
• Both DAF & CD59, import to protect RBCs against bystander lysis.
• Hereditary angioedema (HAE): recurrent attacks of swelling that affect the extremities, gastrointestinal tract, skin, and other mucosal surfaces- this known to be complement deficiency disorder.
• Population frequency is 2 in 10,000, since caused by C11-INH defiance.
• C1-INH's was named: controlling complement for the contact pathway of the coagulation system that is critical in HAE.
• C1INH: serpin (serine protease inhibitor, that contacts/controls number for serine proteases)
• The absence of C1-INH results localized swelling that can be either subcutaneous or found bowel/upper-respiratory tract. Typically, this occurs in area of oropharynx -> life threatening, due to the edemas that subsides in spontaneous/ 48-72 hours.
• HAE: Separates two types Type 1 -> Decrased C1-INH protein Type 2 normal levels -> Decrease function.
• Generic Cause is both -> autosomal dominant gene & this codes both a dysfunctional/an inactive protein.
• There are acquired forms with that result either: Auto antibodies that will block C1-INH function or of C1-INH consumption
• To differentiate acquired & hereditary form -> Helpful measurement of C1. C1q is Low acquired form/ not in hereditary
• C4 is also to a helpful screening for HAE particularly during attack- the patients does not show C4 at the drop.

HUS (Hemolytic uremic syndrome) Factors

• Hemolytic uremic syndrome (HUS) that has Renal deficiency in Children, with characterized with hemolytic anemia, low platelet count, and has Anemia of renal deficiency too.
• The Shiga toxin relates to infections w/acute the dirrehea & the reason primary: The HUS -Atypical HUS is an (aHUS), It occurs - Complement dysregulation due/to the polymorphys- genetic.
• Atypical HUS -> Is/ isn't be associated has less acute onset and the it is dirrehea Clinical presentation is -Similar.
• Genetic mutations in HUS -> Factors B,Factor H, MCP. Factor I and with the auto antibodies of thrombomodulin. -> The/w Factor H & Factor I.
• Complement has also be been implicated w/ C3 Glomerulopathies , which it is disease that involves / w C3 glomerulopathies (C3G), diseases that involve the glomeruli of the kidneys.
  • The rare glomerulonephritis (GN) with forms- severel w/reclassified the Presence /Absence of C3 w/ immunoglobulins, based Immunoflurescences.
  • To C3- it's found only 3, C3G (Glomeruropathies).

C3 (Nephridic Factors)

• The study of patients shows -> 71/per -> Mutation in Proteins, has, (100%), C3, Factor H, B, and I.
  • Another have antibody acquiring -> It known auto C'3 (aka. Nephilitic Factors. /C3NeF.)
    • An antibody :Binds C3's convertase ( from Path/Alternative)-> A holding all together with mechanisms controls will make it's resistance with of, C3bbb.
      • In C3NeF--> The leading of uncontrol (CleAVAGE) - C3 with -> Controll the position's over Kidneys
      • C3G by all -> A CLIN-indistinguishable, to disorder's form hereditation. Laboratory:
• Measured by laboratory: Nature's, (disorder's) -> A measuring only w and/absence the C'3NeF
• Investigating C3NeF for : Possible -> In deficiency - complection Components. > Be consumed activate
• Auto antibodies/relate activated Consumption <-A key of test laboratory shows required forms- Hered /Compl. for - D.

Immunologic Assays of Individual Components

• Test/measures of complement levels, can diagnose disorder
• Deficiencies of Hereditary + Inflammation and -> States Immune that are learned

• Following. Is the Proteins consumed.
• Methods and /techniques - Determinate abnormalities = Two Categories

• Measurement of components " Serum and/as
  • Activity for functional Measurement.
• For test (RID - > Nephemet) use for. Each/individual use commonly. Turbidity tests :C3 /C4 Level measurements.

A Nephemetry/by-Turbity routine "Clinical Lab test- automations can show that.

Explore the roles of CR1, MCP, and DAF in complement regulation. Understand how these proteins contribute to self/non-self discrimination and prevent autoimmune reactions. Learn about anaphylatoxins C3a and C5a and their effects on immune cells.