In the classical pathway, C1 is the first enzyme in the cascade. It binds to the Fc region of antigen-complexed antibody molecules. It is a pentamolecular molecule made up of one C1q, two C1r and two C1s, and is Ca2+ dependent. Activation is initiated by the binding of C1 to complexed antibody. The first step is the binding of antibody to two or more of the six globular domains of C1q. This then binds to the complexed IgG or IgM causing changes in conformation of the C1 complex. This leads to the activation of one of the C1r molecules by autocatalysis, then the other to produce to active C1r enzymes. These enzymes then cleave the two C1s molecules to produce two active C1s serine esterases. C1 then cleaves C4 and generates activated C4b which at this point is unstable. It can either be hydrolysed by water to form inactivated iC4b or can form covalent bonds with amine or hydroxyl groups on cell surface molecules to form surface bound C4b, which can now act as a binding site for the zymogen C2. The bound C2 is a substrate for C1s and is cleaved to producing C2a and C2b. C2b is released, whereas C2a remains bound to form C4b2a, the C3 convertase enzyme.
In the alternative pathway, the binding of C3b to a protected surface, is followed by an amplification step that results in the binding of many more molecules of C3b to the same surface. The bound C3b binds to Factor B to produce C3bB by way of an Mg2+ dependent bond. Factor D, a serine esterase, then cleaves factor B to release Ba, leaving C3bBb bound to the surface. The C3bBb dissociates fairly rapidly unless it is stabilized by the binding of properdin (P), forming the complex C3bBbP. This constitutes the surface bound C3 convertase enzyme of the alternative pathway.
The final phase of activation of the complement cascade applies to both pathways. It is the formation of the MAC by enzymatic cleavage of C5, a protein homologous to C3 and C4; however, it lacks the internal thioester bond. In order for it to be cleaved, it must be bound to C3b. The C5 convertase enzyme for both pathways are trimolecular. In the classical pathway, the complex is C4b2a3b, where the c3b is covalently bound to the C4b. In the alternative pathway, the complex is C3bBb3b, in which one C3b is covalently bound to another. Cleavage of C5 releases C5a, a potent anaphylatoxin. C5b binds to C6 forming C5b6 which then binds to C7 creating a C5b67 complex. Once C7 has bound, the complex changes from hydrophilic to hydrophobic, hence inserts itself into lipid bilayers. C8 then adds on followed by the addition of up to fourteen C9 monomers, leading to the formation of a pore forming molecule. The MAC has a hydrophobic external face, allowing it to associate with the lipid membrane bilayer, but a hydrophilic internal channel. This channel allows the free passage of solutes and water across the lipid bilayer. The disruption of the lipid bilayer leads to the loss of cellular homeostasis eventually leading to lysis. When C8 binds, a small amount of lysis occurs, but the majority occurs after the binding of C9. This causes osmotic disruption, hence lysis of the target cell. Therefore if the pathway goes to completion, lysis will occur.
The pathways of the complement system
RESULTS
Size of pellets:
Tube 1 = Largest
Tube 2 = Fairly large
Tube 3 = Small
From the graph(s), it can be seen that the amount of haemolysis occurring in each of the three tubes does vary. This is due to the contents of each one. Tube 1 displays very little haemolysis, as it only occurred in the most concentrated well, i.e. the 1 in 4 dilution, and no haemolysis occurred thereafter. Tube 2 shows more haemolysis than in tube 1 as it occurred in the first three wells, but the amount of haemolysis steadily decreased with dilution until by the fourth, there was none. Tube 3 showed the greatest amount of haemolysis as complete haemolysis occurred in three of the twelve wells and partial haemolysis in a further three wells.
Tube 1 follows the classical pathway as its complement activator was bovine serum albumin-anti-bovine serum complex, an antibody-antigen complex.
After centrifuging the mixture in tube 1, a fairly large pellet was formed. The pellet contains all the bound complement, which is likely to constitute the C1 molecule due to it’s a molecular mass, as C1q is 460kD, C1r is 157kD and C1s is 150kD. The supernatant is likely to consist of the unbound complement including C3, as it has a much lower molecular mass of only 174kD therefore would not settle and form the pellet. So, the supernatant contains the necessary components to carry the pathway on some of the C3 convertase has been made to cleave the C3 hence continue. The C1 is no longer required as it has performed its role by producing the C3 convertase which would have occurred during the incubating period.
The more concentrated the complement is, i.e. well 1, the more lysis is likely to occur. In the case of tube 1, very little lysis occurred. As can be seen from the graph, there is no 100% endpoint, only a 50% endpoint, therefore I shall be adopting the 50% endpoint for all tubes. This is the point at which half of the cells present haemolyse, which in this case is slightly greater than 0.25µl. This means that from the dilutions carried out, none of them performed complete haemolysis. This may be due to the fact that a lot of the complement was bound, therefore a high molecular weight, hence the large pellet, so there was not a lot of complement left in the supernatant for the pathway to complete, therefore cannot produce the MAC. Due to lack of complement, the wells 2-12 were unable to haemolyse.
Tube 2 follows the alternative pathway. This is known as it does not contain an antigen-antibody complex. It is the zymosan, a component of yeast cell walls that initiates this pathway. Following centrifuging the tube, the pellet formed was quite large, but not as big as that found in tube 1. As explained above, the pellet contains all the bound complement as it would have a greater molecular mass. This would have had more unbound complement, hence more haemolysis occurring as is shown from the results. In this case there a 50% endpoint of approximately 0.094µl. This shows that the difference in concentration for haemolysis to occur by half, is quite small. After 0.25µl, only partial haemolysis takes place until it reaches a concentration of 0.03125µl.
The reason for why there is greater haemolysis in tube 2 compared to that of tube 1 is due to the binding of C3b to the antigenic surface. In mammalian cells, there is a high level of sialic acid. This rapidly inactivates any bound C3b molecules on the host cell. Foreign antigenic surfaces, such as a yeast cell wall, have low levels of sialic acid, so the C3b that binds to these surfaces remain active for a longer period. The C3 convertase activity of C3bBb has a half life of only five minutes unless the serum protein, properdin, binds to it, hence stabilizing it, extending its half life to thirty minutes. The C3 convertase produced in this pathway is able to activate unhydrolysed C3 to produce more C3b autocatalytically, resulting in the initial steps being repeated and amplified. So that more than 2 ×106 molecules of C3b can be deposited on an antigenic surface in less than five minutes. This can then go on to produce the C5 convertase and continue the pathway leading to MAC formation, hence lysis.
Tube 3 does not represent any of the complement system pathway as it is a standard control so the results of this tube can be used to compare both the classical and alternative pathway, hence is crucial for the experiment. The 50% endpoint for this tube is 0.0078125. This tube shows that as the concentration gets more dilute, there is less lysis occurring. The pellet formed after centrifugation was very little which shows that there was little bound complement in the tube. All the unbound complement remains in the supernatant therefore it is this that is used in the process. Therefore a lot of C3 will be present so can form MAC, hence cause a lot more lysis. As it becomes more dilute, less complement is present, therefore the pathway will not complete so no lysis will occur.
Erythrocytes are poorly lysed by homogenous complement, but readily lysed by complement derived from other species. Two proteins have been characterised that mediate this species restriction. CD59 is a protein anchored by a glycophospholipid foot. It is widely distributed in cell membranes, binds to C8 in C5b-8 complexes and inhibits the insertion and unfolding of C9 into cell membranes. A second protein, homologous restriction factor (HRF) has similar activity to CD59 but is probably a weaker inhibitor of C9 insertion. Nucleated cells, such as cell’s of the host’s immune system, are much more resistant than erythrocytes to lysis by complement, because they can actively remove MAC by endocytosis and exocytosis of fragments of membrane containing MAC.
The results show that the activators used both showed some haemolysis, however, that of tube 2 appears to be more efficient as it showed a better trend of results. They both clearly displayed the effects of each of the two pathways.
The technique used was in all relatively successful as my results show where haemolysis occurred and gave me the expected results.
The experiment may have led to errors, which may be due to
- Insufficient mixing of reagents
- Unequal volumes added to wells
- Pipetting errors
In order to ensure accuracy of results, the procedure could have been repeated.
References
Immunobiology – The Immune System in Health and Disease 4th edition (1999)
Charles A Janeway et al
Published by current Biology publications
Immunology 4th edition (1996)
Roitt et al
Published by Mosby
Instant notes in Immunology (2000)
P. M. Lydyard et al
BIOS Scientific Publishers Ltd.
