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MBW:Immune Complement

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A summary of The Alternative Pathway of Complement by Michael K. Pangburn and Hans J. Muller-Eberhard[1] by Matanya Horowitz. An in depth investigation of the results is done in a later project.

Background

  • The human body has a large battery of defense mechanisms used to protect itself against foreign infectious agents. Among these is the complement system, a biochemical cascade of reactions that is part of the body's innate defense system. The complement system is divided up into two pathways, the classical pathway and the alternative pathway. It is the alternative pathway that is the focus of this paper.
  • In the body, the liver produces the base protein of the complement system, C3. These C3 proteins can break apart spontaneously in water into two smaller proteins, C3b and C3a. (For information on modeling the formation of C3b as a Poisson process, please see APPM4390:Complement Modeling). This C3b, as well as another protein, Factor B, can then serve as catalysts for this reaction. If a pathogen is present in the body, C3b can bind to the membrane of the pathogen. If this occurs the C3b, while attached to the pathogen membrane, can bind with Factor B, to form the protein C3bB. This new complex is then cleaved into two proteins designated Ba and Bb. Bb remains attached to C3b on the surface of the pathogen, to form C3bBb, also called C3 Convertase, while Ba floats off.
Figure 1: Complement alternative pathway overview. Generated using SimBiology Matlab package.[2]
  • The C3 Convertase, now attached to a pathogen, serves as a catalyst and rapidly breaks apart free floating C3 proteins into C3a and C3b. This renews the detection cycle and results in a positive feedback loop that deposits large quantities of C3bBb on the surface of the pathogen.
  • The presence of C3 Convertase and C3b also allow for a larger structure, C3bBbC3b to form. This protein can split apart another protein of the complement system, C5, into C5a and C5b. These proteins initiate a cascade of reactions that result in C6, C7, C8, and C9 proteins forming a large structure together, called the membrane attack complex (MAC). The MAC complex forms on the surface of a pathogen and once it is large enough, it 'punches a whole' into the pathogen. This allows for lysis of the pathogen as the the whole thats created by the MAC allows for free diffusion to occur from between the inside and the outside of the pathogen.
  • This paper focuses on what is labeled the Preparation, Regulation, and Amplification portions of the alternative pathway in Figure 1.

Introduction

  • The alternative pathway is made up of six primary plasma proteins that are used in recognition and activation. These proteins remain in the body and provide a continuous surveillance mechanism. An important aspect of complement is that it does not require specific antibodies, and can therefore attack a variety of pathogens. The body regulates these proteins and prevents the activation of the pathway against its own cells through the use of regulatory plasma and membrane proteins.
  • The primary proteins used for initiation and for positive feedback amplification of the pathway are C3, Factor B, and Factor D. To regulate, Factor H, Factor I, and Properdin are used.
  • The goal of the pathway, once activated, is to deposit large numbers of C3b molecules and to construct the cytolytic membrane attack complex (MAC). Once the MAC has been created the pathogen is destroyed through the free diffusion created by the MAC as well as by other components of the immune system that are triggered.

For more information about the immune cell's role, see APPM4390: Mathematical Immunology.

Activation

Preparation

Figure 2:Deposition of C3b (indicated by fluorescence) on pathogen surface over time[1]
  • The source of the alternative pathway is the C3 protein. In an aqueous solution, this protein hydrolyzes to form C3(H2O), which shares many properties with C3b. Among these is the property that it can be split by Factor D and bound with Factor B, producing C3(H2O),Bb(Mg) and releasing the fragment Ba. This 'tick over' mechanism allows for consistent low levels of C3b to exist in the body, providing a method for foreign particle detection.
  • C3 may also be split into C3a and C3b by either C3b itself or C3(H2O),Bb(Mg). This exposes a highly reactive metastable binding site on C3b which provides for the binding of C3b with carbohydrates, a typical component of pathogenic membranes. This theory is supported by experimental evidence showing strong ester bonding between C3b and zymosan, a yeast cell wall polysaccharide.

Detection

  • Figure 3: Distribution of fluorescence (C3b) on pathogens over time[1]
    Figure 4: Distribution of fluorescence (indicating C3b concentration) on pathogens with consistent initial distribution[1]
    Initially, C3b bonds to carbohydrates present on pathogen membranes randomly, simply attaching as the C3b floats near the pathogen. These deposits of C3b then induce greater production of C3b which can then rapidly cover the pathogen's surface. This theory is supported by experimental evidence, as illustrated in Figures 2 and 3. Figure 2 illustrates an initial lag period from when initial deposits are made until large amounts of C3b begin to cover the pathogen's membrane. This lag is a characteristic of an initially small random distribution self-reinforcing and instigating a positive feedback loop. Furthermore, this causes asymmetry when viewing the fluorescence distribution over time in Figure 3. If the distribution had not been random, the results would have been a more gradual increase in fluorescence without asymmetry. This was verified by initiating a large number of cells with low levels of C3b, removing the variability of the initial deposits. The results may be seen in Figure 4 in which the fluorescence of the cells are seen to be distributed along a simple bell curve at each point in time, matching expectations.

Recognition

  • Due to the binding flexibility of C3b, without protection activation would occur on human body cells as well. To prevent this, host cells have a number of proteins that prevent C3b formation on cell membranes. Among these are Factor H and Factor I that are present in the body. These proteins inactivate C3b that has become bonded to the cell membrane, preventing the C3b from activating the positive feedback loop. This has been experimentally verified by comparing rabbit erythrocytes, which don't have these proteins, to sheep erythrocytes, which do have the proteins. As expected, sheep erythrocytes successfully resist destruction by C3b while the rabbit erythrocytes do not. However, introduction of Factors I and H to rabbit erythrocytes inhibited but did not stop C3b formation, indicating that other proteins contribute to recognition.

Membrane-Associated Regulation

  • The primary method by which the body prevents autoimmune attacks on the part of complement is through proteins embedded in cell membranes. These proteins, CR1 and decay accelerating factor (DAF) prevent C3b formation through inactivation and by encouraging the decay of membrane embedded C3b.
  • CR1 binds to C3b on the surface of the membrane and acts as a cofactor with Factor I to cleave C3b, removing it from the membrane. It can also bind with deactivated C3b, C3bi, breaking it into C3c and C3dg. It also serves to inhibit the binding of C5 to C3b. However, cells that lack CR1 have been observed to be able to successfully defend themselves against C3b, indicating that while CR1 holds a prominent role in preventing C3b formation, it is not the decisive regulator.
  • DAF behaves similarly to CR1 in its ability to prevent C3b formation on the membrane. However, studies have shown that the inhibition of DAF severely limited the cells ability to fight off C3b formation, indicating that DAF is the primary regulatory membrane associated regulator.

Paroxysmal Nocturnal Hemoglobinuria

  • An acquired and unfortunate disease known as Paroxysmal Nocturnal Hemoglobinuria (PNH) has the effect of creating deficiencies in an individual's DAF and CR1 production. This has allowed for research into the alternative pathway to investigate which of these proteins has the greater effect on complement defense. The results of these studies indicate that only DAF is required to protect a cell from complement. CR1 appears to assist in the defense, but by itself it isn't sufficient. The exact role of CR1 remains unknown.

Amplification

Figure 5: Amplification reactions on pathogenic membrane surface[3].
  • Once C3b has bound to the surface of a pathogenic membrane it has the potential to form C3 convertase. This reaction requires Factors B, D, and Mg++ and results in the protein Ba being released. C3 convertase then serves as an enzyme to cleave C3 into C3a and C3b, which can then in turn bind to the membrane, creating the positive feedback loop.
  • The Properdin protein stabilizes the C3 convertase and serves to boost the amplification of bound C3b. Properdin isn't a necessary component to the alternative pathway in most cases. However, there are a few situations, such as in the defense against measles, wherein the amplification that Properdin provides allows for lysis of infected cells. Without Properdin, the infected cells are able to clear the C3 convertase sufficiently quickly to defend themselves. The aforementioned reactions can be seen graphically in Figure 5.
  • The remaining aspects of the alternative pathway that result in the destruction of the pathogen, namely the formation of C5b and the MAC, were not investigated in this paper, but are described in the overview.

References

  1. 1.0 1.1 1.2 1.3 Pangburn, Michael K. & Muller-Eberhard, Micheal J. "The Alternative Pathway of Complement". Spring Seminars in Immunopathology. 163-192
  2. Morley, Bernard J. & Walport, Mark J (2000). The Complement Facts Book p. 88. Academic Press. ISBN 0-12-733360-6.
  3. Rantes. http://en.wikipedia.org/wiki/File:Droga_alternatywna.png