26 Complement System-II

Dr. M. N. Gupta

 

  1. Objectives
  •  To understand MBL pathway of complement activation.
  •  To explain the Doughnut hypothesis for the membrane attack complex action.
  •  To understand other biological effects of the complement.
  •  To understand how complement action is regulated.
  1. Concept Map
  1. Description

Let us start with looking at the 3rd activation pathway in which activation of complement is initiated by mannan binding lectin binding to mannans on the cell surface of some infectious bacteria and fungi.

 

MBL pathway

 

This is yet another way the complement activation is initiated.

 

In this pathway, initiation is by binding of the host plasma protein MBL which is called mannose binding lectin or mannan binding lectin. This pathway is homologous to the classical pathway. MBL belong to collectin family. These are pattern recognition proteins and can bind to bacterial and fungal pathogens whose surface has mannose or similar sugar residues.

 

Collectins are defined as complement components which include MBL, conglutinin and soluble pattern recognition receptors. These all can activate complement. While vertebrate cell surfaces also have these sugar residues, these are masked by terminal sialic acid residues. MBL deficiency in infants is associated with proneness to respiratory infections.

 

Innate immune response is important during the period before the child‟s adaptive immune responses have matured and passive immunity from maternal antibody via placenta or colostrums is lost.

 

MBL is present at low concentration in plasma of most individuals. The acute phase reaction of the innate immune response leads to ~3-fold increase in the production of MBL by liver.

MBL resemble C1q in its appearance of “bunch of tulips”. The six “heads” have a stalk which has structure similar to collagen. The “heads” are the carbohydrate binding sites.

 

Lectins are not catalytic in nature. These proteins just bind to carbohydrates in free form or as a part of glyconjugate structures. In this case the stalk of MBL is associated with two series proteases MASP-1 and MASP-2 (MASP stands for MBL associated serine protease)

 

MASP-1 and MASP-2 are similar to C1r and Cls which reflects the analogous nature of MBL and C1q. The pathogens to which MBL is known to bind includes Salmonella, Listeria, Neisseria sp, Candida albicans and Cryptococcus necoformans. Once MBL is bound to the surface of the pathogens, MASP get activated and cleave C4 and C2. This forms C3 convertase from C2B bound to C4B as in the classical pathway. This lectin pathway also can get activated by ficolins binding to bacterial carbohydrates

 

Ficolins are a group of three lectins which recognise bacterial cell wall and apoptotic cells.

 

Self Vs Non self

 

Complement action is a part of innate immunity. The alternative pathway is not dependent upon antibody molecule. When adaptive immune responses evolved, complement action also evolved to create classical pathway. The lectin pathway is also similar to classical pathway. Hence, if innate immunity was the only and hence important defence mechanism, can it also distinguish between self and nonself?

 

The key step is the covalent binding of C3b to particle like microbes. C3b functions like opsonin and allows attack by membrane attack complex. The C3 activation occurs on the surface of the microbe and not in the plasma or self cell surfaces. Covalent binding of C4b occurs to the microbe surface. This happens after C4 cleavage exposed a reactive thioester bond on the C4b.

 

We had learnt that C4 cleavage is initiated by C1. Similarly in MBL pathway, MBL bound to the pathogens surface initiated this.

 

C4b reach with adjoining molecules on the microbe surface. If this does not happen, the thioester is hydrolysed by water and inactivates C4b.

 

So, active C4b cannot diffuse away and attack self cells. Same is the case with C3b formed subsequently.

 

Also, self cells also have molecules which prevent C3b deposition.

 

Relationship between pathways of complement activation

 

It is important to understand the close relationship before 3 pathways of complement action.

 

The divergence between the three pathways is primarily at the initiation steps.

  •  In classical pathway C1 binds to the invading microbe directly (innate immunity purely) or to the antibody bound to the antigenic surface (which involves adaptive immunity)
  •  In MBL pathway, MBL binding to microbe activates MASP activity.
  •  In the alternative pathway, this function is carried out by factor D.

 

The doughnut hypothesis

 

It is Burton D. Goldberg of the N. Y. University School of Medicine and Howard Green of MIT who showed that when cells are attacked by this complement, they swell and get ruptured spilling out the contents. A cell membrane damaged by the complement behaves like a semipermiable membrane. Salt and water flow in according to Donnan effects.

 

One question which puzzled scientists was that does the destruction of the cell requires multihit (many holes) with a threshold value? Alternatively, all it may require is a single hole in which the number the number of destroyed cells directly depend upon the amount of the complement.

 

Manfred M. Mayer and others had carried out experiment which supported one hit theory.

 

It was also found that >5 MAC and probably even one or two are enough to destroy a single cell. Liposomes (concentric lipid bilayers with enclosed small molecules) have been useful in further understanding of the complement action. Complement was found to damage the lipid bilayer. An old hypothesis had been the leaky patch hypothesis according to which complement gives rise to leaky patch. A typical leaky patch (with no rigid structure of a hole) should be able to change in size over time.

 

Electron microscopy showed that lesions on the membrane produced by the complement were uniform. In fact, while guinea pig complement produced 8.5-9.5nm internal diameter holes, human complement produced holes of 10-11 nm internal diameter.

 

The hole persist for about 30 min, which led Meyer to propose doughnut hypothesis.

 

The stable hole is an assembly of a rigid doughnut shaped channel which connects the cellular interior with the extracellular milieu.

 

Subsequent studies have confirmed that complement protein themselves are part of this doughnut like channel.

 

Other biological effects of complement

 

MAC causes cell lysis of pathogens. In addition to that complement also participate in other biological processes.

 

Amano and Inone of the University of Osaka, Japan had showed that in the case of gram negative bacteria, serum lysozymes and complement act synergistically.

 

Phagocytosis

 

Phagocytosis is a key process by which immune system defends the body against bacterial infections. Without participation of antibody and/or complement, phagocytes cannot adequately cause cell lysis.

 

In 1950‟s Nelson showed that binding of C3 on cell surfaces resulted in their phagocytosis.

 

The role of the complement is especially important during the early days of bacterial infection when antibody secretion is yet to take off. Wood jr, Winkelstein and Shin at Johns Hopkins showed that alternative pathway (which is independent of antibody) gets activated during this phase. Alper and Rosen at Harvard medical school studied the consequences of defective C3 in many patents and found pronounced susceptibility to pus-producing bacterial infections.

Many cells have receptors for complement components. C3b is a major opsonine but other complement components also can function as such. Table shows the receptors on various cell lysis and the corresponding complement components. Receptors on diverse cells shows that the involvement of complement may not be totally restricted to immune response.

 

It may be added that binding of C3b on the surface of microbe is not enough to induce phagocytosis. Phagocytes also have receptor for C5a whose binding starts phagocytosis.

 

Local inflammatory response

 

Fragment C3a, C4a and C5a are small fragments which are created during complement activation pathways. These can attach to some cells to produce inflammation.

 

This is normally local inflammation. However, if these are produced in large amounts, the result is a generalized circulatory collapse called anaphylactic shock. This can be induced in experimental animals by injecting large amounts of these complement fragments systemically. Hence, their fragments are called anaphylotoxins.

 

C3a and C5a can activate submucosal populations of mast cells to release histamine and TNF-α. C5a and C3a mediate recruitment of antibody, complement and phagocytes to the site of infection. Increased fluid accumulation in the tissue facilitate the pathogens carrying antigen presenting cells to move to lymph nodes which initiates vigorous adaptive immune response. C5a directly acts on neutrophils and monocytes to enhance their adherence to vessel walls, and their phagocytic action (Figure 8).

The C-termini of both C3a and C5a contain an arginine residue which is essential for their anaphylatoxin action. Carboxypeptidase which can specifically remove this is present in serum. Hence this enzyme is called Anaphylatoxin inhibitor. However removal of Arg does not inhibit their chemotaxis action.

 

Complement mediated chemotaxis

 

Different complement components differ in the nature of their targets. C5a not only attracts neutrophils, it stimulates their respiratory burst and aggregates them.

 

The biological consequences of complement are summarized in the following figure

 

Complement system does not participate in immune defense mechanism alone. It also interacts with another important phenomenon related to blood-blood clot formation which is in fact another cascade phenomenon.

Plasmin, which is a protease can activate both C1 and C3 and is inhibitor by C1 inactivator

 

C1 inhibitor can also inhibit thrombin, factor XI and factor XII, plasmin and kallikrein..

 

C3b can cause platelet aggregation. This leads to release of ADP which in turn results in release of vasoactive factors like histamine and serotonin. Platelet procoagulants are released which accelerate the clot formation.

 

It has been reported that rabbits congenitally deficient in C6 show poor blood clotting tissue.

 

Regulation of the complement

 

Two inherent features of complement system are designed to ensure that complement does not attack „self‟ cells of the host. All complement components occur as inactive molecules.

 

The activation takes place on pathogen surface. Unless, the components bind, these are inactivated spontaneously. For example the target binding site on C3b gets hydrolysed during the time it takes to diffuse away by 40 nm from the C4b2a or C3bBb. Nevertheless, complement is activated slowly in the blood spontaneously albeit at a very slow rate. Also, as always there are back up regulatory features to ensure that complement does not damage self cells of the host. Such regulatory controls primarily operate either in the beginning or towards the end of the activation pathways. All the 3 pathways are regulated.

 

Defects in these regulatory controls are known to lead to serious diseases. The basic feature of an important regulatory control is shown below

C1 inactivator (C1INH)

 

It is the key regulator of the classical pathway. Glycoprotein of 105kDa, it controls the assembly of classical C3 convertase by blocking C1r and C1s.

 

Its congenital deficiency produces excess C2 kinin. Resultant increase in vascular permeability leads to tissue edema (especially around mouth and then on face and neck) Possibly larynx swelling leads to suffocation of the patient. This disease is known as hereditary angioedema.

 

Five other glycoproteins which regulate C3b and C4b are factor H, C4-bp (plasma protein) and membrane protein CR1, decay accelerating factor (DAF), and gp 45-70. All are members of the same multigene family.

 

DAF and gp 45-70 have similar activity with gp 45-70 showing a preference for alternative pathway convertase whereas DAF can bind to both classical or the alternate convertases.

 

Neutrophils can destroy MAC by endocytosis or exocytosis. The membrane vesicles containing MAC are either shed or ingested and destroyed. This represents yet another illustration of diverse mechanism by which host self cells can escape complement.

Figure 13: Complement activation is regulated by a series of proteins that serve to protect host cells from accidental damage.

 

It seems that initially complement developed as a part of innate immunity and then evolved to work in synergy with adaptive immunity. Even now, in the early days of infection before adequate antibody secretion starts, complement mounts the defence against the microbe.

 

This is one clear example where an early system did not fade away but evolved to retain its utility.

 

Summary

 

  •  Mannan binding lectin (MBL) can also initiate complement activation.
  •  All the pathways differ in initiation steps.
  •  Operation of Donnan effect during MAC action is explained by the Doughnut hypothesis.
  •  The other biological effects of complement include opsonisation and producing inflammatory responses.
  •  Complement action is highly regulated to prevent attack on self cells. Many proteins are part of these regulatory mechanism.