complement and complement deficiency

Complement and complement deficiency
Boonthorn
4 September 2009

Outline
Complement pathway activation
Classical pathway
Alternative pathway
Lectin activation pathway
Membrane attack complex
Complement receptors and biological functions
Regulation of complement activation
Disorders associated with complement deficiency
Management of complement deficiencies

Complement system
Important part of innate immune system
Major effector mechanism of humoral immunity
Named by Jules Bordet ( 1919 , Nobel Prize )
Group of serum protein ( normally inactive ) ,activated through sequential protease- based step (under particular condition )
Complement activator, Complement regulatory protein and Complement receptor

Functions of complement. The major functions of the complement system in host defense are shown. Cell-bound C3b is an opsonin that promotes phagocytosis of coated cells (A); the proteolytic products C5a, C3a, and (to a lesser extent) C4a stimulate leukocyte recruitment and inflammation (B); and the MAC lyses cells (C).

Pathways of Complement Activation
3 major pathways of complement activation
classical pathway : activated by Ab bound to Ag
alternative pathway : activated on microbial cell surfaces in the absence of antibody
lectin pathway : activated by plasma lectin that binds to mannose residues on microbes
central event in complement activation is proteolysis of C3 to generate biologically active products and subsequent covalent attachment of C3b to microbial cell surfaces or to Ab bound to Ag
Abbaset.al.Cellular&Molecular immunology 6th edition

Overview of the complement pathways indicating components required for recognition, enzymatically active components and complexes, major opsonic, inflammatory and membranolytic products.
Rich et al.Clinical immunology principle and practice. third edition.

The early steps of complement activation by the alternative and classical pathways.The alternative pathway is activated by C3b binding to various activating surfaces, such as microbial cell walls, the classical pathway is initiated by C1 binding to antigen-antibody complexes, and the lectin pathway is activated by binding of a plasma lectin to microbes. The C3b that is generated by the action of the C3 convertasebinds to the microbial cell surface or the antibody and becomes a component of the enzyme that cleaves C5 (C5 convertase) and initiates the late steps of complement activation. The late steps of all three pathways are the same (not shown here), and complement activated by all three pathways serves the same functions

Alternative pathway
Activation : proteolysis of C3
C3 tickover
C3b , binding site for factor B covalently tethered to surface of microbial or host cell
Bound factor B cleaved by factor D (serine protease)
C3 convertase : C3bBb
Properdin bind to and stabilize C3bBb complex
C5 convertase : C3bBb3b

Internal thioester bonds of C3 molecules.A schematic view of the internal thioester groups in C3 and their role in forming covalent bonds with other molecules is shown. Proteolytic cleavage of the α chain of C3 converts it into a metastable form in which the internal thioester bonds are exposed and susceptible to nucleophilic attack by oxygen (as shown) or nitrogen atoms. The result is the formation of covalent bonds with proteins or carbohydrates on the cell surfaces. C4 is structurally homologous to C3 and has an identical thioester group.

The alternative pathway of complement activation.Soluble C3 in plasma undergoes slow spontaneous hydrolysis of its internal thioester bond, which leads to the formation of a fluid-phase C3 convertase (not shown) and the generation of C3b. If the C3b is deposited on the surfaces of microbes, it binds factor B and forms the alternative pathway C3 convertase. This convertase cleaves C3 to produce more C3b, which binds to the microbial surface and participates in the formation of a C5 convertase. The C5 convertase cleaves C5 to generate C5b, the initiating event in the late steps of complement activation

Classical pathway
Activate :IgM>IgG3>IgG1>IgG2>>IgG4
C1 : large, multimeric, protein complex
C1q binding site in Cμ3 (IgM),Cγ2 (IgG) domain through its globular head group
C1r, C1s subunits are proteases
Activated C1s cleaves C4, to generate C4b
C2, complexes with C4b and cleaved by C1s to generate soluble C2a and larger C2b fragment
C3 convertase : C4b2b
C5 convertase : C4b2b3b
C5a : potent inflammatory activity
C5b initiate formation of MAC

Structure of C1.C1q consists of six identical subunits arranged to form a central core and symmetrically projecting radial arms. The globular heads at the end of each arm, designated H, are the contact regions for immunoglobulin. C1r and c1s form a tetramer composed of two c1r and two c1s molecules. The ends of c1r and c1s contain the catalytic domains of these proteins. One c1r2s2 tetramer wraps around the radial arms of the c1q complex in a manner that juxtaposes the catalytic domains of c1r and c1s.

C1 binding to the fc portions of igm and igg. C1 must bind to two or more fc portions to initiate the complement cascade. The fc portions of soluble pentamericigm are not accessible to C1 (A). After igm binds to surface-bound antigens, it undergoes a shape change that permits C1 binding and activation (B). Soluble igg molecules will also not activate C1 because each igg has only one fc region (C), but after binding to cell surface antigens, adjacent iggfc portions can bind and activate C1 (D).

The classical pathway of complement activation.Antigen-antibody complexes that activate the classical pathway may be soluble, fixed on the surface of cells (as shown), or deposited on extracellular matrices. The classical pathway is initiated by the binding of C1 to antigen-complexed antibody molecules, which leads to the production of C3 and C5 convertases attached to the surfaces where the antibody was deposited. The C5 convertase cleaves C5 to begin the late steps of complement activation.

Lectin pathway
Activation : D-mannose,GlcNAc ( microbial polysaccharides )
Binding to circulating lectins, eg. Mannose -binding lectin (MBL), or to N-acetylglucosamine recognizing lectins (ficolins ) collectin family and structurally resemble C1q
MASPs eg. MASP-1, 2, and 3 (tetrameric complex similar to C1r and C1s )
MASP-1 cleave C3
MASP-2 cleave C4 and C2
C3 convertase : MASP-1
C5 convertase : C4b2b3b

Activation of the lectin pathway. the lectin activation pathway is engaged by pathogen oligosaccharides (black diamonds ) and leads to a C3 convertase which is identical to that of the classical pathway. MBL serves as the recognition domain of the complex ,while MASP1 and MASP are similar in function to C1r and C1s
Middleton’s allergy:principles and practice. 7th edition.

Membrane attack complex
Cleavage of C5 into C5a and C5b.
C5 (structurally homologous to C3 and C4, lacks internal thioester bond )
C5b initiates formation of MAC (complex of C5b, C6, C7, C8 and multiple C9 molecules ) binds to C6, and C7 , recruits C8 and complex penetrates more deeply into the membrane.
C9, a pore-forming molecule with homology to perforin. The complex of C5b678 forms a nidus for C9 binding and polymerization
Penetrates membrane bilayers to form pores
Disrupt the osmotic barrier, leading to swelling and lysis of susceptible cells

Late steps of complement activation and formation of the MAC.Cell-associated C5 convertase cleaves C5 and generates C5b, which becomes bound to the convertase. C6 and C7 bind sequentially, and the C5b,6,7 complex becomes directly inserted into the lipid bilayer of the plasma membrane, followed by stable insertion of C8. Up to 15 C9 molecules may then polymerize around the complex to form the MAC, which creates pores in the membrane and induces cell lysis. C5a released on proteolysis of C5 stimulates inflammation.

A. Complement lesions in erythrocyte membranes are shown in this electron micrograph. The lesions consist of holes approximately 100 Å in diameter that are formed by poly-C9 tubular complexes. B. For comparison, membrane lesions induced on a target cell by a cloned CTL line are shown in this electron micrograph. The lesions appear morphologically similar to complement-mediated lesions, except for a larger internal diameter (160 Å). CTL- and NK cell-induced membrane lesions are formed by tubular complexes of a polymerized protein (perforin), which is homologous to C9 C. A model of the subunit arrangement of the MAC is shown. The transmembrane region consists of 12 to 15 C9 molecules arranged as a tubule, in addition to single molecules of C6, C7, and C8 α and γ chains. The C5bα, C5bß, and C8ß chains form an appendage that projects above the transmembrane pore.
Structure of the MAC in cell membranes

Receptors for Complement Proteins
CR1 or CD35 :
On erythrocyte,neutrophil,monocyte,eosinophil,T and B cell
Bind C3b,C4b and promote phagocytosis
Opsonization ,activate phagocyte when Fc receptor engaged by Ab coated particle
clearance of immune complexes from circulation
Regulator of complement activation
CR2 or CD21 :
On B lymphocyte, FDC
Bind C3d,C3dg and iC3b
enhancing B cell activation ( CR2,CD19,TAPA-1 orCD81)
promote trapping of Ag-Ab complexes in germinal centers (FDC)
Receptor for EBV virus

RECEPTORS FOR COMPLEMENT PROTEINS
CR3, CD11bCD18 or Mac-1 :
On neutrophil,mononuclearphagocyte,mast cell, NK cell
Bind iC3b promote phagocyotsis,ICAM-1 promote attachment to endothelium
CR4, p150/95, CD11cCD18 :
on dendritic cells
binds iC3b
function probably similar to Mac-1

Receptors for Complement Proteins
The complement receptor of the immunoglobulin family (CRIg)
on macrophages ( Kupffer cells )
integral membrane protein
bind C3b and iC3b
clearance of opsonized bacteria and other blood-borne pathogens
SIGN-R1
C-type lectin on marginal zone macrophage
recognizes pneumococcal polysaccharides
bind C1q, activation of classical complement pathway in an antibody-independent manner
opsonization and clearance of pneumococci

Regulation of complement activation
C1 inhibitor
Regulators of the C3 and C5 convertases
Factor I
Soluble regulatory proteins; C4b-binding protein and factor H
Membrane regulatory proteins, CD55 (DAF), CD46 (MCP), CD35 (CR1)
Properdin
Regulators of the MAC
Soluble MAC inhibitors; S protein, and clusterin
Membrane MAC inhibitor; CD59

C1 inhibitor
C1 inhibitor (C1-INH)
serine proteinase inhibitor (serpin) , SERPING1
binds to activated C1r and C1s, limit classical pathway activation.
inhibits MASP-1 and 2, kallikrein, factor XIa, XIIa and plasmin of the lectin pathway and the contact, coagulation, and fibrinolytic systems.
( Autosimal dominant ) inherited deficiency of C1-INH is basis of hereditary angioedema , plasma level of C1 inh<20-30% of normal

Serpin inhibitory mechanism. Active serpin(pink) is characterized by a 5-stranded b-sheet (b-sheet A, cyan) and an exposed reactive centre loop (RCL, green)presenting the P1 residue to the active site of the target protease (gold)(a). The initial step is the binding of free protease and active serpin RCL (b). Subsequently, the protease cleaves the RCL. RCL cleavage triggers a profound conformational change within the serpin molecule: the RCL downstream of the scissile bond inserts betweenstrands 3 and 5 of b-sheet A as strand 4, while the protease is translocated to the opposite pole of the inhibitor molecule and deformed, resulting in enzyme inhibition by active-site distortion (c).Therefore, protease-serpinacyl-enzyme complex formation can be viewed generally as biologically irreversible.
Trends in Molecular Medicine Vol.15 No.2

Regulation of C1 activity by C1 INH.C1 INH displaces C1r2s2 from C1q and terminates classical pathway activation

C3b bound by membrane cofactor protein ( MCP ,CD46), CR1, decay-accelerating factor (DAF), factor H
C4b bound by DAF, CR1, C4-binding protein (C4BP)
competitively inhibit binding of other components of C3 convertase, eg. Bb (alternative pathway ) and C2b ( classical pathway ) , blocking further progression of the complement cascade
Factor H inhibits binding of only Bb to C3b

The regulation of the classical pathway. The classical activation pathway is shown with the relevant regulatory proteins shown in the gray hexagons. C4 BP denotes C4 binding protein

The regulation of the alternative pathway. The alternative activation pathway is shown with the relevant regulatory proteins shown in gray hexagons. C3bBb is stabilized by properdin.

Inhibition of the formation of C3 convertases. Several membrane proteins present on normal cells displace either C2b from the classical pathway C3 convertase (A) or Bb from the alternative pathway C3 convertase (B) and stop complement activation.

DAF
glycophosphatidylinositol-linked membrane protein
on endothelial cells and erythrocytes.
If deficiency , Causes paroxysmal nocturnal hemoglobinuria.
unusual feature , acquired mutation in defective gene ,in hematopoietic stem cells.
factor I
plasma serine protease degrade cell-associated C3b
active only in presence of regulatory proteins
MCP, factor H, C4BP, and CR1 serve as cofactors
cause dissociation of C3b (and C4b)-containing complexes
Generates iC3b, C3d, and C3dg, recognized by receptors on phagocytes and B lymphocytes

Factor I-mediated cleavage of C3b. In the presence of cell membrane-bound cofactors (MCP or CR1), plasma factor Iproteolytically cleaves C3b attached to cell surfaces, leaving an inactive form of C3b (iC3b). Factor H and C4-binding protein can also serve as cofactors for factor I-mediated cleavage of C3b. The same process is involved in the proteolysis of C4.

Regulation of formation of the MAC
CD59
Membrane protein inhibit formation of the MAC
glycophosphatidylinositol-linked protein expressed on many cell types.
incorporate into assembling MACs after the membrane insertion of C5b-8, thereby inhibiting the subsequent addition of C9 molecules
present on normal host cells, where it limits MAC formation, but it is not present on microbes
S protein
binding to soluble C5b,6,7 complexes
Prevent insertion into cell membranes near the site where the complement cascade was initiated

Regulation of formation of the MAC. The MAC is formed on cell surfaces as an end result of complement activation. The membrane protein CD59 and S protein in the plasma inhibit formation of the MAC.

COMPLEMENT DEFICIENCIES
Genetic deficiencies in classical pathway components
Deficiencies in components of the alternative pathway
Mutation of the gene encoding the mannose-binding lectin (MBL)
Deficiencies in the terminal complement components
Deficiencies in complement regulatory proteins
Deficiencies in complement receptors

C1q deficiency
Present with early onset SLE , anti-dsDNA less common ,more severe disease , less steroid responsive
Strongest known genetic risk factor for lupus
C4 deficiency
C4A deficiency , risk factor for SLE (less severity than sufficient complement) early onset SLE , anti-dsDNA less common ,
C4B deficiency, with invasive bacterial disease

C2 deficiency
most common of the inherited classical complement component deficiencies in Caucasians
most common cause of death : sepsis
most common organisms :S. pneumoniae and H. influenzae
C3 deficiency
rarest of the four early component deficiencies
most severe phenotype
Membranoproliferativeglomerulonephritis
neutrophil dysfunction (abscesses), humoral deficiencies (sinopulmonary disease), and complement deficiencies (sepsis, meningitis).

FACTOR B DEFICIENCY
single case has been reported ( meningococcemia )
FACTOR D DEFICIENCY
Neisserial infections are the most common manifestation
PROPERDIN DEFICIENCY
X-linked complement deficiency
one of the more common complement deficiencies and occurs largely in Caucasians
one or more episodes of meningococcal disease
high fatality rate in contrast to terminal complement component deficiencies

GUPTA et al.Indian J Med Res 127, May 2008, pp 431-440

MBL DEFICIENCY therapy
Plasma derived MBL is now being produced by Co-operative Research Centre for Vaccine Technology (CRC-VT)
on phase I safety, tolerability and pharmacokinetics
MBL replacement therapy used in 3 situations.
where MBL deficiency leads to increased susceptibility of infection, increase resistance to that infection
In acute infection,may enhance the resolution of the disease
Used to alter the natural history of chronic disease
GUPTA et al.Indian J Med Res 127, May 2008, pp 431-440

Deficiencies in the terminal complement components

Deficiencies in complement regulatory proteins

C1 inhibitor deficiency
Hereditary angioedema
mildly increased susceptibility to infection
increased risk of SLE
Recurrent episodes of angioedema, involvement of airway in absence of anaphylaxis, abdominal episodes, a positive family history, or angioedema arising after trauma
Type I deficiency ,most common (85%) of inherited cases
Type II deficiency normal production ,but dysfunctional protein

C1 inhibitor deficiency
main manifestations of C1 inhibitor deficiency are recurrent episodes of submucosal or subcutaneous edema
severity of episodes do not correlate with laboratory features
extremities, face, or genitalia are most often involved
The most feared type of angioedema is that involving the airway
angioedema typically progresses for 1–2 days and resolves in another 2–3 days. Common precipitants are illness, hormonal fluctuations, trauma, and stress

Contact system

pathogenesis of angioedemadue to C1-INH deficiency. In HAE, the deficiency of C1-INH is due to a mutation in the C1-INH gene, which impairs C1-INH synthesis or function. In AAE, C1-INH deficiency is due to the cleavage of C1-INH by autoantibodies or to its consumption by neoplastic, mainly lymphoproliferative, tissue. Reduced C1-INH plasma levels result in hyperactivation of the classical complement pathway with increased consumption of C1-INH and further reduction of its plasma level. Impaired inhibition of activated Factor XII (FXIIa) and kallikrein, as a result of a lack of their principal inhibitor C1-INH, enables cleavage of high molecular weight kininogen (HK) by kallikrein and release of bradykinin, which binds to its B2 receptors, causing edema
Tissue permeability
vasodilatation

Acquired C1 inhibitor deficiency
clinically indistinguishable from inherited
that the age of onset is after 30 years
The laboratory features are similar to those of hereditary C1 inhibitor deficiency except that C1q levels are diminished in these patients
B cell malignancies and monoclonal gammopathies are the most common

Trends in Molecular Medicine Vol.15 No.2

CD59 DEFICIENCY AND PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
PNH :
recurrent episodes of hemoglobinuria due to intravascular hemolysis
Thrombosis occurs for unknown reasons
aplastic anemia
due to acquired somatic mutations of PIG-A or PIG-M in a clone of bone marrow progenitor cells
These protein products are required for GPI-anchored proteins and C8-binding protein, DAF and CD59 are all GPI-anchored proteins which protect hematopoietic cells from complement-mediated lysis
DAF deficiency does not have a hemolytic phenotype
CD59 is more important

Deficiencies in complement receptors

CR3/CR4 deficiency
β2-integrin deficiency is leukocyte adhesion deficiency type I (LAD type I)
Mutations in the common chain (CD18)
LAD type I is very serious disorder with high mortality rate
Very high resting wbc counts, frequent necrotic skin infections without pus formation, delayed separation of umbilical cord, and assorted other serious bacterial and fungal infections
bone marrow transplantation is recommended ( severe case )

conclusion
Middleton’s allergy:principles and practice. 7th edition

J ALLERGY CLIN IMMUNOL . APRIL 2004

Thank you

complement and complement deficiency

by Division of Allergy and Clinical Immunology, Department of Medicine, Faculty of Medicine, Chulalongkorn University on Sep 07, 2009

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complement and complement deficiency — Presentation Transcript