The complement system, the blood clotting system, the clotrlysis system, and the bradykininkinin system constitute the major protein mediator pathways of the bloodstream. In fact, they are all closely related and activation of one may lead to some activation of them all. The complement system is often thought of as a single entity when it really consists of 20 or more proteins that interact precisely with one another. Another common misconception is that the complement system exists solely to punch holes in cell membranes and thus provide an independent method for the destruction of cells, bacteria, viruses, and fungi. This concept arose from the original description of the actions of complement as it was defined by its ability to lyse sheep erythrocytes in the test tube. The activities of the complement system are considerably more complex than cellular destruction, although that capability is certainly important. In general, the activities of the complement system may be divided into the three categories below (Table 1).
Cell Lysis. Activation of the complement system may lead to formation of the membtane attack complex (MAC), possibly resulting in cell membrane damage associated with loss of ability to maintain an osmotic grathent, thus causing lysis of cells. Activation of either the classical or the alternative pathway of the complement system may be responsible for this outcome.
Inflammation. Activation of the complement system results in the generation of small peptides which mediate anaphylaxis, Chemotaxis, and vasodilatation. These factors may also promote degranulation of mast cells and neutrophils, causing the release of other mediators that augment the inflammatory response. Larger proteins of the complement system may coat activating particles or immune complexes. This may lead to enhanced phagocytosis (opsonization) as a beneficial result, or may facilitate deposition of the particles or complexes in tissues. This deposition causes increased inflammation and organ damage, due to both the primary effects of complement activation and the secondary influx of neutrophils and mononuclear phagocytes (MNPs) called to the area by the chemotactic stimuli.
Immunological Effects. Activation products of the complement system play important roles in both B and T cell-mediated reactions. For instance, C3 is necessary for the localization of antigen within germinal centers and the subsequent production of antibody. Antibody responses to T cell dependent but not T cell independent antigens are suppressed by depletion of the complement system, with C3 probably being the most important reactant. C5a enhances primary antibody responses in vitro although it is currently believed that the cause of this enhancement is related to C5a's effects on the production and secretion of interleukin 1 by the MNPs. Activation peptides of C3, C5, factor B, and factor H also influence MNPs to promote inflammation and immunologic reactivity.
COMPLEMENT SYSTEM ACTIVATION
COMPLEMENT SYSTEM PATHWAYS
Rather than concentrating on the details of specific reaction sequences, this article will outline some of the more recent concepts of the mechanisms and activities of the complement system.
PATHWAYS OF THE COMPLEMENT SYSTEM
Although the exact workings of the complement system involve a series of complicated and, as yet not fully understood molecular events, an understanding of this system's function may be obtained through a description of the concepts underlying these events. The reaction sequence of the complement system may be separated into four main divisions: the classical pathway, the alternative pathway, the terminal pathway or membrane attack complex, and the feedback amplification loop (Table 2). The classical pathway consists of those proteins that mediate recognition. activation, and inhibition relating primarily to specific antibody binding. The alternative pathway is defined by those proteins that provide recognition, activation, and inhibition when exposed to repeating polysaccharide structures such as those found on bacterial or fungal cell walls. This is clearly a more primitive and less specific response. If activation of either the classical or alternative pathway is allowed to proceed then the mechanism of terminal pathway or membrane attack complex may be set in motion. This pathway is common to both the classical and alternative pathways and once activated, results in the formation of open channels through cell membranes that prevent the maintenance of an osmotic grathent and hence cause osmotic lysis. The fourth pathway, the feedback amplification loop, allows for the rapid generation of activated complement fragments the role of which is to magnify all of the previously noted complement functions.
Recent investigations have begun to elucidate the important regulatory functions of complement receptors on the cell surfaces of neutrophils, erythrocytes, lymphocytes, and mononuclear phagocytes. Our understanding of the expression and function of these receptors is in an embryonic stage, but it is clear that many in vivo activities of the complement system may be mediated through cellular receptors. This may be an important fifth division of the complement system.
Complement activation results in two families of molecules: those whose purpose is to promote continued activation of the complement system, and those whose main purpose is to signal biological activities. Complement activation is accomplished by the generation of complexes of complement components that have enzymatic activity. The cleavage of subsequent complement proteins results in either new complexes with new enzymatic activity to continue the activation sequence, or protein fragments with biological activities. Interestingly, some components of the complement system belong to both families, playing an important role in continuing the complement sequence as well as having crucial biologic activities.
Central to an understanding of the complement system is the recognition that concurrent with system activation is also system inhibition. This inhibition takes both an active and passive form. The passive form is described as "decay/dissociation," which means that the activities of enzyme complexes and active fragments spontaneously decay and lose their enzymatic or biological activities. Active inhibition occurs through the activities of control proteins. These proteins may displace one of the proteins in a complex by competing for a binding site, they may bind to an enzymatic site thus neutralizing it, or they may enzymatically cleave a protein thus inactivating it. The expression of the effects of complement activation are therefore dependent on two rates: the rate of activation and the rate of inactivation. If there is an overwhelmingly persistent activating stimulus or a deficiency in the control proteins, the net result will be significant complement activation and the biologic effects noted above - cell lysis, inflammation, and immunologic activation. On the other hand, if the capacity to inhibit or degrade exceeds the rate of stimulation, a marked damping of the reactions will ensue and very different biologic results will occur.
Both the classical and alternative pathways have the common goal of producing an enzyme capable of cleaving C3 into the two fragments, C3a and C3b (Figure). These enzymes are called C3 convertases. This cleavage is the critical step because addition of more C3b molecules to these enzymes results in enzymes that can cleave C5 into C5a and C5b. The fragments, C3a and C 5 a, are the main anaphylatoxins that are responsible for inflammation. The complements C3a, C5a, C3b, and C5b are fragments that provoke immunologic responses. C5b is the starting point for assembly of the membrane attack complex. As expected, the inhibitory proteins concentrate their activities on this enzyme complex as well.
Historically, the classical pathway was recognized first, but since the alternative pathway is simpler and probably appeared earlier in phylogeny, it will be discussed first.
The Alternative Pathway
The alternative pathway consists of four proteins related to activation: properdin (P), C3, factor B, and factor D; and two proteins related to inactivation: C3b inactivator (I) and BjH (H). Complement receptor 1 (CRl) seems to play the same role as H. It is currently accepted that due to structural instability, there is a low-grade, continuous alteration of C3. By binding to this C3b-like molecule, factor B becomes susceptible to the action of factor D which cleaves a small peptide from factor B, Ba. The remaining large piece, Bb, now has the capability of splitting C3 into C3a and C3b. The released C3b will bind to new factor B, allowing factor B to be activated. This escalating reaction would rapidly proceed to consume all of the available C3 were it not for the activity of H which displaces Bb from C3b. C3b, not in complex with Bb, is quite susceptible to cleavage by I, resulting eventually in the biologically inactive C3c and C3d and cessation of complement activation. Properdin acts to stabilize the enzyme, C3bBb, and may prolong its half-life as much as tenfold. The half-life of this alternative pathway enzyme may also depend upon the nature of the activating substance or particle. Both the quantity of sialic acid and the amount of decay-accelerating factor present on the surface of cells determines their ability to support or prevent the assembly of the alternative pathway enzymes on their surfaces.
ASSESSMENT OF COMPLEMENT SYSTEM
The Classical Pathway
Classical pathway activation occurs after antibody has complexed with antigen. This binding induces a conformational change within the Fc fragment of the immunoglobulin molecule that allows for specific interaction with the first component of the complement system. Although components in the classical pathway are numbered, the numbers were assigned in order of discovery and not by functional order. Thus, the classical pathway consists of six components: CIq, CIr, CIs, C2, C3, and C4. The inhibitors in this system consist of Cl esterase inhibitor, C4 binding protein (C4bp), and I. CRl may act similarly to C4bp in this system as well. CIq forms a calcium-dependent complex with CIr and CIs. Upon binding to complexed immunoglobulin, a number of internal cleavages result in the activation of CIs. Both C4 and C2 are the natural substrate for CIs resulting in the functional fragments C4b and C2a. If C4b binds either to the antigen or to the antibody, C2a may complex to it. This complex of C4b and C2a, C4b2a, is the classical pathway C3 convertase and will generate both C3b and C3a from native C3. This complex may be dissociated by C4bp with subsequent cleavage and inactivation of C4b by I as well as spontaneously by decay/ dissociation.
The Membrane Attack Complex
Both the C3 convertase of the alternative pathway and the C3 convertase of the classical pathway become C5 convertases by binding additional molecules of C3b. These C5 convertases cleave C5 to C5a and C5b. Once C5b becomes bound to a surface, it assembles the rest of the membrane attack complex without further enzymatic activity. Molecules of C6, C7, C8, and C9 form a functional unit capable of disrupting cell membrane integrity using C5b as their point of attachment. This reaction is independent of all previous complement reactions and may occur on cells unrelated to those that provoked complement activation, thus called "bystander lysis." Since there are no direct inhibitors of this sequence, once C5b is generated, the reaction proceeds to completion. Effective inhibition can occur only at the stage of C3 and C5 convertases and not after C5b is generated (Figure 3).
Figure. Pathways of complement activation.
Activation in both the classical and alternative pathways depends upon the presence of surfaces. In the classical pathway, antibody binds to a surface; in the alternative pathway, C3b binds to a surface. However, C3b in the fluid phase may also participate in complement activation using the alternative pathway components, factors B and D. C3b forms complexes with factor B allowing factor B to be cleaved by factor D. This results in a fluid phase C3 convertase which generates more C3b and then more enzyme. This complex is inhibited by BjH and I as in the alternative pathway.
DEFICIENCIES OF COMPLEMENT PROTEINS
ASSESSMENT OF THE COMPLEMENT SYSTEM
There are three types of measurements that may be useful in assessing the complement system (Table 3). The first demonstrates the quantitative presence of the individual proteins. We now recognize that the quantity and structure of each complement protein is determined by genes, half inherited from the mother and half from the father. In general, quantitative values are established using immunochemical techniques because antibodies specific for each complement protein may be raised in rabbits or goats. Both heterozygous and homozygous deficiencies may be recognized using these quantitative techniques. The second type of measurement tests the functional activity of the proteins. For instance, in hereditary angioedema caused by Cl esterase inhibitor deficiency, 15% of patients have a genetically determined inactive form of this inhibitor protein despite adequate quantities as determined by immunochemical techniques. Tests have been devised that analyze each component's activity using hemolysis as the end point. The last type of measurement probes specifically for activated complement components. C3a, C4a, C5a, and Ba may be separated from their larger parent molecules by physical means and then quantitated by radioimmunoassay or enzyme-linked immunoassay. Activation complexes may also be detected in biological fluids using combinations of specific antibodies. A complete analysis of a patients complement system must therefore take into account synthetic capability (quantity), functional activity and activation status (consumption) of both the classical and alternative pathways.
As one might expect from the above noted functions of the complement system, assessment of the complement system or individual components is indicated in a wide variety of clinical situations (Table 4). Deficiencies of the components of the classical pathway have often been associated with autoimmune diseases, while deficiencies of the membrane attack complex components may be responsible for recurrent infections with neisserial organisms. Deficiencies of C3 or C5, because of their central role in complement activation, are often associated with recurrent infections by encapsulated organisms, but may also be part of some autoimmune syndromes. It is essential to remember that low levels of complement components may be due to consumption, and it is necessary to consider activation as a factor whenever complement component levels are low. Such situations may arise during sepsis, autoimmune disease, and exposure to the membranes of hemodialyzers or membrane oxygenators.
The activities of the complement system encompass cell destruction either directly through the membrane attack complex or indirectly via the effects of inflammation. The effects of complement fragments on immune functioning are just now being elucidated. Recent advances in methodology have allowed recognition of complement deficiencies of both quantitative and structural varieties. Complement activation studies may detect subclinical activation and be useful guideposts for therapeutic intervention. Coupling our new understanding of the mechanisms of complement activation with new technologies to measure this activation may result in the better understanding of the pathology of inflammation and its concurrent immunologic reactions.
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Complement Assays: Cooper NR, Nemerow GR, Mayes JT: Methods to detect and quantitate complement activation. Springer Semm lmmunupothol 1983; 6:195-212.
Complement Pathways: Porter RR, Reid KBM: Activation of the complement system by antibody-antigen complexes: The classical pathway. Adv Protein Cfiem 1979; 33:1-71.
Gotze O, Miller Eberhard HJ: The C3 activator system: An alternative pathway of complement activation. J Exp Med 1971; l34(suppl):90S-108S.
Complement Receptors: Ross GD, Medof ME: Membrane complement receptors specific for bound fragments of C3. Adi' Immunol 1985; 37:217-267.
COMPLEMENT SYSTEM ACTIVATION
COMPLEMENT SYSTEM PATHWAYS
ASSESSMENT OF COMPLEMENT SYSTEM
DEFICIENCIES OF COMPLEMENT PROTEINS