The complement system

Discuss the mechanisms by means of which defects in components of the complement system can lead to a variety of human diseases.

1.1 Introduction

The complement system is the major effecter of the humoral branch of the immune system which is present before birth. The components are synthesized in the foetus by around 3 month gestation period and the components are activated before 25 weeks in the developing foetus.[1] It consists of about 30 serum and membrane proteins whose biological activities affect the innate and adaptive immune systems. They account for about 5% of the plasma protein. The complement proteins are mainly synthesized by the liver hepatocytes while a small percent is synthesized by the blood monocytes, macrophages and epithelial cells of the gastrointestinal and gastrourinary cells.[2]

The complement activation can occur by 3 different pathways -

1. Classical Pathway - C1 is composed of C1q and 2 units of C1r and C1s. When an Ag-Ab complex binds to the C1q subunit the CP is activated. This C1 complex further leads to proteolysis of C2 and C4, whose larger fragments bind to form the C3 convertase.

2. Lectin Pathway - Mannose binding Lectin (MBL) analogous to C1q binds to certain bacterial mannose moieties. MBL associated serine proteases 1 and 2 (MASP's) are structurally similar to C1r and can activate the lectin complement pathway and lead to the formation of C3 convertase.[3]

3. Alternative Pathway - This pathway leads to amplification of the complement system and is initiated by foreign cell-surface constituents. Factor B (which is analogous to C2 in CP) binds to serum C3b, Factor D and Properdin which cleaves Factor B, and the resulting component C3bBb has C3 convertase activity.

The C3 convertase initiates a cascade of events forming the C5 convertase which leads to the formation of the Membrane Attack Complex C5b6789 (MAC) which inserts itself into the surface of the target cell, forming a large channel which allows ion exchange and thus leading to cell lysis.

The main functions of the complement are opsonisation, immune clearance, lysis of cells, bacteria, viruses by the MAC, some of the smaller fragments of the complement cascade act as anaphylotoxins (C3a and C5a) which can mediate a powerful inflammatory response. The threshold for B cell activation is lowered when an antigen is co-presented with the complement component C3d via CR2.[4]

1.2 Regulation

The complement system is highly regulated as it has a high potential to create injury in host cells and tissues if it is misdirected or over activated. Thus the complement molecules are controlled by regulators and inhibitors at each important level.[5]

Fluid Phase regulators - these regulators are distributed in the plasma and consist of Factor H which regulates amplification of the AP, properdin, Factor I, C1 inhibitor which is a CP and LP regulator, inhibitor of the terminal pathway CFHR1 and clusterin.[6]

Membrane bound regulators - includes regulators like CR1 which induces dissociation of C3 convertases of the CP and AP and also recruits Factor I, decay acceleration factor and MCP which is a co-factor for factor I and protectin which protects the cell from non-specific damage of host cells by the MAC by binding to C8 on the host cell membrane.[7]

Receptors for complement effector molecules - There are five major complement effector receptors which are membrane bound CR1, CR2, CR3, CR4 and CRIg to which C3b and C4b bind to carry out their corresponding biological activities. C3a and C5a bind to C3aR and C5aR respectively which then mediate inflammatory responses.[8]

C4bp is a regulator which is attached to the cell surface which targets Factor I to C4b and thus the formation of the C3 convertase is blocked.

2 Complement Deficiencies

Complement deficiencies are generally rare and many of the complement disorders are specific to a certain populations. Complement related diseases can arise due any deficient component in any of the 3 pathways or defects in complement regulation which occurs in a hierarchy.

All the three complement pathways converge at the point of C3 convertase formation and thus regulation at this stage is perhaps the most important. Secondly the regulation at this level for the alternative pathway is also extremely important as it is the major amplification loop. This is done by Factor H and thus I will elaborate on FH deficiencies due to it's key role in the complement pathways regulation and also FH deficiency can lead to diseases which although rare, can lead to a great extent of renal injury and can be life threatening.

2.1 Factor H

Factor H is a single polypeptide chain glycoprotein which is composed of 20 repetitive units of 60 amino acids named SCR's. The gene encoding Factor H (CFH) is a member of the RCA on chromosome 1q32 with CFH haplotypes CFH1 - CFH5. Factor H is mainly synthesized in the liver and also in a cell types like retinal epithelial cells, peripheral blood lymphocytes, myoblasts, mesengial cells etc.[9]

The main function of factor H is to down-regulate the alternative pathway which it does in the 3 ways, firstly it acts as a co-factor in the factor I mediated proteolytic cleavage of plasma C3b, secondly Factor H binds to C3b thereby preventing the binding of component B and lastly Factor H promotes the dissociation of the C3 convertases i.e. possesses decay acceleration activity.[10]

Thus FH deficient patients show low plasma concentration of C3 and high concentration of C3b due to uncontrolled activation of the alternative pathway.

Factor H deficiency is associated with mainly 3 clinical conditions -

2.11 MPGN

“Membranoproliferative glomerulonephritis (MPGN) also termed as dense deposit disease is an uncommon cause of chronic nephritis characterized by proliferation of mesangial and endothelial cells and by thickening of the peripheral capillary walls due to sub endothelial immune and/or intramembranous dense deposits” i.e. deposition of C3 and C9 within the glomerular basement membrane due to the unrestricted activity of the AMP i.e. hypocomplementaemia and affects mainly children and young adults.[11] Thus the glomerular basement membrane thickens which impairs filter function, hematuria, proteinuria and eventually leads to loss of renal function.[12]

According to study on mice, pigs and humans MPGN has only been observed in homozygous (CFH-/-) mutants and no histological evidence of MPGN has been seen in heterozygous (CFH+/-) mutants.[13] This FH deficiency is due to mutations in the SCR regions of the CFH genes.

One strain of pigs develop rapidly progressive MPGN when deficient in factor F due to a mutation in the SCR20 in which the mutant protein expressed but is retained in the endoplasmic reticulum and thus it accumulates in the cell cytoplasm. This leads to complete renal failure in about 4 weeks. FH deficient mice which were developed by targeting gene deletion of exon 3 in the embryonic stem cells also developed features of MPGN within 8 months.[14]

Mutation in the CFH gene, in the regions CCP 9 and CCP 16 affect disulphide bond formation when there is an absence on cys residues in the conserved framework, any amino acid exchange in CCP2, CCP7 or in CCP11 result in non-functional FH protein which then may lead to MPGN.[15]

In a study two patients were reported with MPGN2 and the functionally defective protein showed a homozygous deletion of the Lys224 in the CCP 4 regulatory region. Genetic analysis of the DNA derived from the two patients was carried out which showed a nucleotide exchange in the promoter region of the CFH gene, deletion of a Lys residue at position 224 and three other nucleotides in the CCP4 region. A single nucleotide exchange was observed in CCP 7, CCP5 and the intronic region between CCP13 and CCP 14. Experiments carried out showed that the deletion of Lys224 on CCP4 region affected the function of the N-terminal of the protein chain. The mutation affected the C3b binding ability of the protein as the mutation is located on a surface-exposed loop between the conserved β-1 and β-2 strands which correspond to a binding site. Cofactor activity of the mutated protein was reduced to about 10% of the wild-type protein and the protein also showed highly decreased decay accelerating activity.[16]

A single case of MPGN has been reported due to a monoclonal Ig Δ antibody by binding to FH.[17] C3NeF an auto-antibody (IgG or IgM) which stabilizes the convertase C3bBb is usually associated with MPGN2, but it does not play an important role in the development of MPGN but increases the damaging potential of the uncontrolled activity of the ACP.[18]

2.12 aHUS

Hemolytic uremic syndrome is a disease can be divided into two types - Typical HUS is nonimmune haemolytic anemia, thrombocytopenia which leads to renal failure due to platelet thrombi in the kidney microcirculation and follows with diarrheal illness. It is triggered by environment factors, drugs or infections like Escherichia coli producing shiga like toxins.[19]

Atypical HUS is due to the deposition and amplification of C3b which binds to the microvasculature cellular surface and results in damage and destruction of tissue due to lack of complement regulation.[20] Most of the cases of HUS mainly occur in childhood but some cases of adult HUS have been found. aHUS is caused due to deficiencies i.e. heterozygous gene mutations coding for Factor H, Factor I and MCP. “Deficiencies in factor H are involved in both autosomal-dominant and autosomal-recessive haemolytic uremic syndrome”[21]

In a study carried out on 16 FH deficient homozygous and heterozygous patients with atypical HUS and MPGN by Durey et al. It showed that partially deficient patients for FH showed half antigenic levels of FH whereas no detectable levels of FH were observed in complete FH deficiency. 10 patients were found to have HUS. 3 patients presented with a nucleotide substitution in the SCR 15 at position 899 and 924 which led to the creation of a stop codon. Another patient showed a single nucleotide insertion at position 2303 in SCR 13 which also led to a creation of stop codon. A nucleotide substitution where cysteine was exchanged for another amino acid was found in four patients in SCR 7, SCR 11 and SCR 15 respectively. One of the patients had a 25 bp deletion in SCR 2 which led to the change in amino acids and led to the creation of a stop codon in SCR 3. Two patients had an exchange of amino acids i.e. Ser for Phe and Leu or Trp respectively in the SCR 20 region. Except two all other patients were heterozygous for the FH deficiency.[22]

The majority of the CFH mutations found in relation with HUS are missense changes that are clustered in the exons which encode the C-terminal domain of the FH protein[23] i.e. SCR 20, which are usually single amino acid substitutions. These substitutions affect the binding of the protein to the C3b components as one of the C3b binding components is within the SCR 16 - SCR 20 region, further the binding site for heparin and heparin like proteins (sialic acid on glycoproteins and glycolipids) are on SCR 7, SCR 19 and SCR 20. All or most of the above mentioned functions are lost in a mutated FH protein.[24] Thus mutated FH proteins express normal regulatory activity but shows reduced activity in cell protection from complement lysis.[25] This leads to dysfunction of the inhibitory effect of FH and thus induces uncontrolled ACP activation which leads to autolytic attack on host cells by deposition of C3b indiscriminately.[26] Further gene conversions or rearrangements between CFH and CFHr1, deletion of genes in CFHr1 and CFHr3 and MCPggaac haplotypes, and mutations in CFI and CFB have been frequently observed in patients with aHUS.[27]

Some level of plasma C3 regulation is required for the development of HUS as compared to complete absence of the protein in MPGN. An experiment was carried out on FHΔ16-20 (SCR 16 - SCR 20 knockout mice). The degree of plasma regulation by C3 was very low but greater than that seen in FH deficient animals (CFH -/- FHΔ16-20 low). Thus it showed that even very low levels of FHΔ16-20 protein was enough to ameliorate MPGN but not enough for the HUS phenotype to develop. In humans aHUS mostly occurs on heterozygous CFH deficient individuals.[28] Thus most of the mutant FH proteins related to aHUS express normally and present normal wild-type co-factor activity for FI.[29]

In an experiment H-deficient mice (FHΔ16-20) did not develop the HUS phenotype spontaneously. The same was observed in non-manipulated FH deficient mice. This showed that HUS is preceded by other factors like drugs, pregnancy, infective agents, cancer therapies etc. along with a CFH deficiency and in humans multiple genetic triggers maybe required to develop HUS.[30]

2.13 AMD

Age related macular degeneration is a leading cause of blindness in the latter stages of life. Individuals with AMD show yellow deposits on the outside of the external retina and retinal pigment epithelium. These yellow extracellular deposits on the RPE are called drusen. This damage to the RPE and outer retina increases the risk of abnormal choroidal neovascularisation of the outer retina i.e. under the macular area which leads to loss of vision.[31]

AMD is a multi-factor disease which is caused due to environment factors like age, lifestyle, smoking, hypertension, family history[32] etc. and it also has a genetic component. The strongest genetic component associated with AMD is the complement factor H gene (CFH) Y420H polymorphism. The most studied SNP is at the CFH locus rs1061170 in the SCR 7 region where there is an amino acid substitution i.e. Tyr is exchanged for His at position 402. The tyr 402 hys substitution also contributes to AMD by affecting the level of inflammation in the outer retina. This mutation in the SCR 7 contains the overlapping binding sites for heparin, the C- reactive protein and alters its binding to glycosaminoglycans and reduces its binding capacity to retinal pigment epithelial cells.[33]

Within the CFH gene there are three polymorphisms in another SNP group, another SNP in exon 11 and 2 intronic SNP's which also show a strong coalition with AMD.[34]

The other complement deficiencies lead to a variety of diseases and an overview of some of those are mentioned below.

2.2 C1- INH Deficiency

The C1 Inhibitor is a regulatory glycoprotein, synthesized in the liver which inactivates the C1 component of the complement system by dissociation of C1r and C1s from C1q. The C1 Inhibitor in encoded by a gene on the long arm of chromosome 11.[35] Individuals with C1-INH show symptoms like subcutaneous swelling, increased vascular permeability, respiratory obstruction of the upper respiratory tract etc. and genetically linked C1-INH deficient individuals develop hereditary angioedema(HAE) or acquired angiedema (AAE).

HAE is an autosomal dominant trait which is due to spontaneous mutations.[36] Studies show that about 20% of individuals with HAE have a RFLP and premature stop codons are created in exon 8 due to single base changes. This results in lack of expression of C1-INH protein.[37]

Another type of HAE is due to the synthesis of a dysfunctional C1-INH protein, which is due to a point mutation in the C1-INH gene. The most studied mutation related to HAE is the Arg 444 location and the frequently observed base substitutions are histidine and cysteine.[38]

AAE is a rare syndrome due to excessive C1 consumption or a C1q specific autoantibody.[39]

Consumption of C1 in patients with AAE is due to the neoplastic lymphatic tissues, mononuclear cells and cells from the pulmonary infiltrate. Many lymphoproliferative diseases like MHUS and NHL are connected with AAE. Recent studies have shown higher risk of developing NHL for people with acquired C1-INH deficiency than the general population. Also anti-C1-INH auto-antibodies consume C1-INH by binding to epitopes near the active centre of the C1-INH protein which leads to destabilisation of the regulator, or boost the vulnerability of C1-INH to C1 cleavage.[40]

2.3 Inherited complement deficiencies

2.31 Properdin Deficiency -

Properdin is a polypeptide which stabilizes the C3 convertase and C5 convertase and thus brings about the amplification of the complement which may be activated by any of the complement pathways. Deficiency in properidin is hereditary and is X-linked. Properdin deficient individuals are highly susceptible to meningococcal disease i.e. mainly Neisseria Meningitidis (serotypes Y and W-135). In an experiment by Ivanovska et al. Properdin deficient C57BL/6 mice were tested by their survival of zymosan induced and LPS induced shock. The C57BL/6 mice were more resistant to the zymosan induced shock as compared to wild type mice which showed greater destruction of end organ function and they also showed higher mortality rate to the LPS shock.[41] Further studies have also shown that properdin deficient individuals are more prone to pneumonia and recurrent otitis media.

2.32 Deficiency in components of the Terminal Pathway -

Individuals deficient in any components of the Membrane attack complex are highly susceptible to neisserial infections. The C6 and C7 genes have been identified on chromosome 5 at the 5p 12-14 MAC 2 gene cluster.C6 deficiency mainly arises due to deletions of single nucleotide in the chromosome which induces a frameshift.[42] C6 or C7 deficient patients usually have extremely low concentrations of the proteins in the serum.[43] The component C8 of the MAC is made of of three subunits - α, β and γ. The α and β subunit genes are closely linked on chromosome 1p32 and the γ genes are on chromosome 9q22.[44] C8 deficiency could be due to the lack of α - γ chain, β subunit or a non-functional β subunit. The C9 gene is on chromosme5p13 and mostly all patients suffering from C9 deficiency are asymptomatic.[45] Individuals deficient in the terminal components mainly suffer from diseases related to gram negative bacteria mainly the Neisseria species, N. meningitidis and N. Gonorrhoea.[46]

2.33 MBL Deficiency -

MBL is the lectin pathway activator and also promotes opsonisation. The MBL-2 gene is located on chromosome 1 position 10q21 and polymorphisms in the promoter region of one of the exons leads to MBL deficiency by disrupting its collagenous region. Around 5% of the population shows MBL deficiency but mainly it is the children who are at risk of bacterial infection[47], mainly pneumococcal infections. Recent studies have shown association of some uncommon respiratory diseases with MBL deficiency and it also increases the risk of SLE.[48]

2.4 C3 Deficiency

The C3 protein in the complement system is required for the activation of the alternative and classical pathways. In humans the C3 protein is encoded by a gene on chromosome 19. The C3 deficiency is hereditary as an autosomal recessive trait. Most C3 deficient patients are susceptible to increased infection by encapsulated pyogenic bacteria while some can also develop autoimmune disorders. The C3 deficiency is usually due to a deletion in the C3 gene which leads to the formation of a stop codon.[49]

C3 deficient patients are also at a risk of increased amyloid β plaque deposition which results in loss of neuronal-specific nuclear protein-positive neurons in the hippocampus and differential microglia activation i.e. neurodegeneration.[50]

In experiments carried out by Ghannam et al. Memory B cells could not be found in C3 deficient patients and also patients showed defects in carrying out adaptive immune responses. “In addition, the development of peripheral CD4+ T regulatory cells following CD3 and CD46 activation in the presence of IL-2 (Tr1) was significantly impaired.”[51] Thus C3 deficiency can lead to the susceptibility to a vast number of infections and autoimmune disorders.

2.5 CR3 Deficiency

CR3 is a heterodimer made up of an α chain (CD11b or CD18) and a β chain[52]. It is found on phagocytes, leukocytes and NK cells. Infected sites show increased expression of CD18 CR3 receptors. Deficiency in CR3 i.e. mainly non-association of the α and β chains which is due to nucleotide substitutions or point mutation in the genes[53] leads to increased bacterial susceptibility. This is due to the incapability of neutrophils to home at inflammatory sites at the time of infection in CR3 deficient individuals. Cr3 deficiency can range from normal cutaneous manifestations to severe infections. Also most patients have shown leukocytosis and the leukocytes from CR3 deficient individuals are deficient in adhesion related functions leading to leukocyte adhesion deficiency.[54]

2.6 DAF, CD59 and CR1 Deficiency

CR1 and DAF are membrane proteins which induce the dissociation of C3 convertase. CD59 binds to C8 on the self membrane of the host cells and prevents binding of C9 to avoid the formation of the MAC. DAF deficiency is due to base substitutions in the gene which leads to the formation of a stop codon. There is also some evidence that CD59 has an autosomal recessive mode of inheritance.[55]

Deficiency in CD59 and DAF is associated with paroxysmal nocturnal hemoglobinuria (PNH) and complement induced cell injury. Recent experiments on mice have also shown that DAF and Crry (CR1 related gene) deficiency leads to macrophage and complement mediated attack on platelets which leads to clearance of platelets from circulation.[56]

3. Treatment

There are various methods of treating MPGN, HUS and AMD which have been observed and studies. The CFH2 haplotype a substitution of Val for Ile at position 62 in the SCR 4 region increases the FH regulatory activity and may lower the risk of the above mentioned diseases by reducing the AP activation. The Ile at position 62 is within the N-terminal C3b binding site which is required for the activity of FI and decay accelerating activities of FH. Two SNP's in CFB and CFH4 haplotype have been found to decrease the risk of AMD.[57]

Other complement deficiencies can be treated in other ways involving different strategies.

In many of the complement deficiencies (e.g. C1 INH deficiency, HUS etc.) the deficient proteins are replaced from blood plasma concentrates. The more indirect therapeutics involve engineering of recombinant therapeutic proteins. In an experiment carried out the Fab antibody arms on an immunoglobulin were replace by regulatory moieties of DAF of CD59 from mice. DAF and CD59 immunoglobulins functioned consistently when activated with enzymes in the presence of a hybrid reagent containing CD59 and DAF.[58]

Monoclonal antibodies have been used which is specific for C5 which prevents the generation of C5b and thus avoiding the formation of the MAC[59]

Many bioengineering strategies have worked in experimental models but have failed to have the same effect in humans. Thus therapeutics for complement deficiencies have a wide scope for research in the future.

4. Conclusion

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