Q. Discuss the pathways leading to complement activation giving examples of diseases where complement regulation is impaired.
The complement system consists of a group of proteins found in the blood plasma and on cell membranes. These proteins play a vital role in assisting the innate and adaptive immune responses with the destruction of pathogens. The main roles of complement are mediating inflammation and killing microorganisms either directly by lysis or indirectly by opsonisation (Ross, 1986).
A number of proteins of the complement system circulate in an inactive state, referred to as pro-proteins. In response to a pathogen, complement proteins are activated by an enzyme cascade whereby an enzyme activates the next enzyme in the pathway leading to a greatly amplified final response.
The complement cascade can be activated via three pathways, and the cause of the activation differs for each pathway. The classical pathway utilises antigen-antibody complexes, the alternative pathway is activated by direct contact with the surface of a pathogen and finally the mannan binding lectin (MBL) pathway is activated by MBL bound on bacteria (Murphy, Travers, Walport, 2008). Each pathway undertakes a unique series of enzyme reactions; however every pathway shares the intermediary step of generating C3 covertase. After this convergence the pathways once again generate differing proteins leading to specific effector actions.
Most complement proteins are made in the liver by hepatocytes and Kupffer cells. Others are manufactured at extra hepatic sites such as in fibroblasts and epithelial cells. These extra hepatic complement proteins probably serve as supplemental complement during local inflammation, provoking an immediate response as opposed to the delay of circulating factors (Colten & Strunk, 1993).
The normal serum concentration of complement proteins vary, ranging from fractions of µg/ml in the case of mannan binding lectin to 1200 µg/ml for C3 (Parslow, Stites et al, 2001). These can be lower in patients suffering from certain maladies such as: malnutrition, angiodema, and glomerulonephritis among others (lab tests online, 2011). Deficiencies of the complement system are most often genetically inherited autosomal recessive conditions.
The Classical Pathway
The classical pathway was the first to be discovered and begins when either a single IgM antibody or several IgG antibodies bound on an antigen themselves bind C1 complex. C1 is a protein complex consisting of a single C1q, and two pairs of C1r and C1s (Figure 1). Proteolytic enzymes are released by C1r which cleave C1s which in turn cleaves C4. Eventually C4b and C2a unite to create C3 convertase. The net result of the classical pathway is the generation of C3a C3b and C5a (Murphy, Travers, Walport, 2008).
C3a generates a local inflammatory response by stimulating mast cells to degranulate and release histamine. Inflammation is a key aspect of innate immunity, important in recruiting granulocytes to the site of infection. C5a incites inflammation in the same manner as C3a as well as having a chemoattractant effect on myeloid cells, particularly effective in drawing neutrophils (Monk et al, 2007). C3b serves both to opsonise pathogens enhancing the action of phagocytes and as an intermediary step in generating C5a.
Deficiencies of the classical pathway
Deficiencies in regulation within the classical pathway are characterised by immune complex disease (Rosen, 1993) and generally confer a minor susceptibility to a range of infections because the other pathways are able to compensate. Problems with initial components C1q, C2 and C4 do cause a serious increase in autoimmune diseases such as systemic lupus erythematosus (Botto, 1998). C1q is important in clearing immune complexes and a lack thereof leads to a deposition of these complexes in glomerular tissue (Berger & Daha, 2007). Individuals with C1q deficiency are also susceptible to infection by encapsulated bacteria.
One example of disease caused by overabundance of complement factors can be seen in acquired C1 inhibitor deficiency. C1 inhibitor normally inactivates C1 (as well as MASP’s 1 & 2 of the MBL pathway) by irreversibly binding to C1r and C1s. Without enough C1 inhibitor, C1 is over expressed resulting in auto activation of complement.
The pathology of hereditary and acquired C1 inhibitor deficiency is angiodema (swelling of blood vessel due to leakage of tissue fluid) and lymphoproliferative disorders such as non Hodgkins lymphoma in the acquired disorder (Castelli et al, 2007).
The Alternative Pathway
The alternative pathway is unique in that it can activate complement by interacting directly with the pathogen surface without the need for an intermediary. This pathway activates by the spontaneous hydrolysis of the thioester bond of C3, which relies on factors B and D to generate alternative pathway C3 convertase and C5 convertase. Two notable regulatory proteins with regards to the alternate pathway are cell surface receptor type 1 (CR1) and factor H. CR1 inhibits the formation of C3 convertase while factor H competes with factor B to accomplish this inhibition. The protein properdin is found in the blood, serving to stay the normally rapid decay of alternative C3 convertase (C3bBb) as well as binding to apoptotic and necrotic cells amplifying local alternate pathway activation (Xu et al, 2008).
Deficiencies of the alternative pathway
Properdin acts as a positive regulator of the alternative pathway, playing a crucial role in the action of C3 on bacterial surfaces by stabilising alternate C3. One such bacteria is N meningitides, and thus individuals lacking properdin do not have stable C3bBb and as such have insufficient alternative pathway activation leading to a greater incidence of meningitis (Agarwal et al, 2010). These infections tend not to be recurrent; this may be because on subsequent infection antibodies which can stimulate the classical pathway are now present. Properdin deficiency is one of the few X-chromosome linked complement deficiencies.
Frequent pyogenic bacterial infections are common in people with alternate pathway deficiencies (Parslow, Stites et al, 2001). There is one reported case of factor B deficiency; the patient in question was suffering from meningitis. Factor D deficiency is also rare, afflicted individuals appear to be susceptible to bacteria of the neisseria genus, though the sparse number of cases make this difficult to state substantively (Biesma et al, 2001).
Factor H is a complement regulatory protein which is crucial in protecting self cells from the alternative pathway. It’s presence on host cells and absence from pathogens directs the complement response, while it also regulates the feedback loop by acting as a co-factor for FI, cleaving C3b to create C3bBb which cannot amplify the complement response (Atkinson & Goodship, 2007). A lack of factor H typically results in damage to renal vasculature, diseases such as haemolytic uremic syndrome and glomerulonephritis. These can be attributed to an overproduction of C3 in the absence of factor H, which then abound in the glomerulus.
The Mannose Binding Lectin Pathway
The mannose binding lectin pathway is activated by using MBL (figure 2) to bind carbohydrates on bacterial and viral surfaces. After binding, MASP-1 and MASP -2 are activated. MASP-2 cleaves C4 and C2 and is both structurally and functionally similar to C1r/C1s of the classical pathway. The action of MASP-1 is not well understood but it is likely to assist in cleavage of C2.
MBL is exiguous in the blood, measuring between 0.002 and 10 ug/ml of serum concentration. Despite its small quantity compared to other complement proteins, absence of MBL is nonetheless linked with recurrent bacterial infections and the development of autoimmune disease. Bacterial infection likely arises as mannose is a common component of Gram negative cell walls and thus MBL would bind to these under normal circumstances.
Deficiencies of the MBL pathway
The MBL pathway is thought to be important during early development. Between around six and eighteen months of age, children have lost the maternal antibodies obtained at birth and have not yet developed a sufficient antibody repertoire of their own. This lack of antibodies hampers classical pathway activation, and many young patients with MBL deficiency present with bacterial infections at this age (Turner, 1991). MBL deficiency is the most common imuunodeficiency, found in some form in one third of Caucasians and around half of African populations.
The affinity of MBL appears heterogeneous for bacteria, some such as S aureus and Candida albicans are bound with high affinity while others such as Streptococcus pneumonia are bound with low affinity (Gupta, Gupta, Harjela, 2008). MBL deficiency appears to have a substantial effect on the likelihood of HIV infection, making one between 3 and 8 times as likely to contract it. MBL is known to bind glycosylated residues on the HIV viral envelope of strains R5 and X4. This binding may in itself neutralise the virus, or otherwise hamper its entry into T cells by other means thus mitigating disease progression (Eisen S et al, 2008).
In summary, the complement system plays an auxiliary role in both the innate and adaptive immune response by: inciting inflammation, forming membrane attack complexes and opsonisation of pathogens. Complements role in inflammation and cell lysis is needed for innate immunity while opsonisation improves the uptake and presentation of antigens by antigen presenting cells which can then better facilitate the activity of the adaptive response. Complement can be activated by one of three pathways, deficiencies in constituents of these pathways can lead to a spate of diseases and ill health.
While complement could be seen as secondary or superfluous, the various conditions brought about by complement deficiencies prove that an intact complement system is required for proper health. Often these deficiencies serve to elucidate or reinforce the true function of complement proteins in the same manner as using knockout mice to observe phenotypic changes when a gene is disabled.
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STRUCTURE OF C1 COMPLEX
STRUCTURE OF MANNOSE BINDING LECTIN