Immunology_Janeway

Immunology_Janeway. Three subfamilies of defensins—α-, β-, and θ-defensins—are distinguished on the basis of amino acid sequence, and each family has members with distinct activities, some being active against Gram-positive bacteria and some against Gram-negative bacteria, while others are specific for fungal pathogens. All the antimicrobial peptides, including the defensins, are generated by proteolytic processing from inactive propeptides (Fig. 2.11). In humans, developing neutrophils produce α-defensins by the processing of an initial propeptide of about 90 amino acids by cellular proteases to remove an anionic propiece, generating a mature cationic defensin that is stored in so-called primary granules. The primary granules of neutrophils are specialized membrane-enclosed vesicles, rather similar to lysosomes, that contain a number of other antimicrobial agents as well as defensins. We will explain in Chapter 3 how these primary granules within neutrophils are induced to fuse with phagocytic vesicles


Immunology_Janeway

Immunology_Janeway. that contain a number of other antimicrobial agents as well as defensins. We will explain in Chapter 3 how these primary granules within neutrophils are induced to fuse with phagocytic vesicles (phagosomes) after the cell has engulfed a pathogen, helping to kill the microbe. The Paneth cells of the gut constitutively produce α-defensins, called cryptdins, which are processed by proteases such as the metalloprotease matrilysin in mice, or trypsin in humans, before being secreted into the gut lumen. The β-defensins lack the long propiece of α-defensins and are generally produced specifically in response to the presence of microbial products. β-Defensins (and some α-defensins) are made by epithelia outside the gut, primarily in the respiratory and urogenital tracts, skin, and tongue. β-Defensins made by keratinocytes in the epidermis and by type II pneumocytes in the lungs are packaged into lamellar bodies (see Fig. 2.6), lipid-rich secretory organelles that release their contents into


Immunology_Janeway

Immunology_Janeway. made by keratinocytes in the epidermis and by type II pneumocytes in the lungs are packaged into lamellar bodies (see Fig. 2.6), lipid-rich secretory organelles that release their contents into the extracellular space to form a watertight lipid sheet in the epidermis and the pulmonary surfactant layer in the lung. The θ-defensins arose in the primates, but the single human θ-defensin gene has been inactivated by a mutation.


Immunology_Janeway

Immunology_Janeway. The antimicrobial peptides belonging to the cathelicidin family lack the disulfide bonds that stabilize the defensins. Humans and mice have one cathelicidin gene, but some other mammals, including cattle and sheep, have several. Cathelicidins are made constitutively by neutrophils and macrophages, and are made in response to infection by keratinocytes in the skin and epithelial cells in the lungs and intestine. They are made as inactive pro-peptides composed of two linked domains and are processed before secretion (see Fig. 2.11). In neutrophils, the inactive cathelicidin propeptides are stored in another type of specialized cytoplasmic granule called secondary granules. Cathelicidin is activated by proteolytic cleavage only when primary and secondary granules are induced to fuse with phagosomes, where it is cleaved by neutrophil elastase that has been stored in primary granules. Cleavage separates the two domains, and the cleavage products either remain in the phagosome or are


Immunology_Janeway

Immunology_Janeway. phagosomes, where it is cleaved by neutrophil elastase that has been stored in primary granules. Cleavage separates the two domains, and the cleavage products either remain in the phagosome or are released from the neutrophil by exocytosis. The carboxyterminal peptide is a cationic amphipathic peptide that disrupts membranes and is toxic to a wide range of microorganisms. The amino-terminal peptide is similar in structure to a protein called cathelin, an inhibitor of cathepsin L (a lysosomal enzyme involved in antigen processing and protein degradation), but its role in immune defense is unclear. In keratinocytes, cathelicidins, like β-defensins, are stored and processed in the lamellar bodies.


Immunology_Janeway

Immunology_Janeway. When αand β-defensins are first synthesized, they contain a signal peptide (not shown); a pro-region (blue), which is shorter in the β-defensins; and an amphipathic domain (AMPH, green–yellow); the pro-region represses the membrane-inserting properties of the amphipathic domain. After defensins are released from the cell, or into phagosomes, they undergo cleavage by proteases, which releases the amphipathic domain in active form. Newly synthesized cathelicidins contain a signal peptide, a cathelin domain, a short pro-region, and an amphipathic domain; they, too, are activated by proteolytic cleavage. RegIII contains a C-type lectin domain (CTLD), also known as a carbohydrate-recognition domain (CRD). After release of the signal peptide, further proteolytic cleavage of RegIII also regulates its antimicrobial activity.


Immunology_Janeway

Immunology_Janeway. A class of antimicrobial peptides called histatins are constitutively produced in the oral cavity by the parotid, sublingual, and submandibular glands. These short, histidine-rich, cationic peptides are active against pathogenic fungi such as Cryptococcus neoformans and Candida albicans. More recently histatins were found to promote the rapid wound healing that is typical in the oral cavity, but the mechanism of this effect is unclear.


Immunology_Janeway

Immunology_Janeway. Another type of bactericidal proteins made by epithelia is carbohydrate-binding proteins, or lectins. C-type lectins require calcium for the binding activity of their carbohydrate-recognition domain (CRD), which provides a variable interface for binding carbohydrate structures. C-type lectins of the RegIII family include several bactericidal proteins expressed by intestinal epithelium in humans and mice, comprising a family of ‘lecticidins.’ In mice, RegIIIγ is produced by Paneth cells and secreted into the gut, where it binds to peptidoglycans in bacterial cell walls and exerts direct bactericidal activity. Like other bactericidal peptides, RegIIIγ is produced in inactive form but is cleaved by the protease trypsin, which removes a short amino-terminal fragment to activate the bactericidal potential of RegIIIγ within the intestinal lumen (see Fig. 2.11). Human RegIIIα (also called HIP/PAP for hepatocarcinomaintestine-pancreas/pancreatitis-associated protein) kills bacteria directly


Immunology_Janeway

Immunology_Janeway. potential of RegIIIγ within the intestinal lumen (see Fig. 2.11). Human RegIIIα (also called HIP/PAP for hepatocarcinomaintestine-pancreas/pancreatitis-associated protein) kills bacteria directly by forming a hexameric pore in the bacterial membrane (Fig. 2.12). RegIII family proteins preferentially kill Gram-positive bacteria, in which the peptidoglycan is exposed on the outer surface (see Fig. 2.9). In fact, the LPS of Gram-negative bacteria inhibits the pore-forming ability of RegIIIα, further enforcing the selectivity of RegIII proteins for Gram-positive bacteria.


Immunology_Janeway

Immunology_Janeway. Summary. The mammalian immune response to invading organisms proceeds in three phases, beginning with immediate innate defenses, then the induced innate defenses, and finally adaptive immunity. The first phase of host defense consists of those mechanisms that are present and ready to resist an invader at any time. Epithelial surfaces provide a physical barrier against pathogen entry, but they also have other more specialized strategies. Mucosal surfaces have a protective barrier of mucus. Through particular cell-surface interactions, highly differentiated epithelia protect against both microbial colonization and invasion. Defense mechanisms of epithelia include the prevention of pathogen adherence, secretion of antimicrobial enzymes and bactericidal peptides, and the flow caused by the actions of cilia. Antimicrobial peptides and the bactericidal lectins of the RegIII family are made as inactive proproteins that require


Immunology_Janeway

Immunology_Janeway. Fig. 2.12 Pore formation by human RegIIIα. Top: a model of the RegIIIα pore was generated by docking the human pro-RegIIIα structure (PDB ID: 1UV0), shown as individual purple and turquoise ribbon diagrams, into the cryo-electron microscopic map of the RegIIIα filament. LPS blocks the pore-forming activity of RegIIIα, explaining its selective bactericidal activity against Gram-positive but not Gram-negative bacteria. Bottom: electron microscopic images of RegIIIα pores assembled in the presence of lipid bilayers. Top structure courtesy of L. Hooper. The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. L. Hooper. The complement system and innate immunity. a proteolytic step to complete their activation, whereupon they become capable of killing microbes by forming pores in the microbial cell membranes. The actions of antimicrobial enzymes and peptides described in this section often involve binding to unique glycan/carbohydrate structures on the microbe. Thus, these soluble molecular defenses are both pattern recognition receptors and effector molecules at the same time, representing the simplest form of innate immunity. The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. The complement system and innate immunity. When a pathogen breaches the host’s epithelial barriers and initial antimicrobial defenses, it next encounters a major component of innate immunity known as the complement system, or complement. Complement is a collection of soluble proteins present in blood and other body fluids. It was discovered in the 1890s by Jules Bordet as a heat-labile substance in normal plasma whose activity could ‘complement’ the bactericidal activity of immune sera. Part of the process is opsonization, which refers to coating a pathogen with antibodies and/or complement proteins so that it can be more readily taken up and destroyed by phagocytic cells. Although complement was first discovered as an effector arm of the antibody response, we now understand that it originally evolved as part of the innate immune system and that it still provides protection early in infection, in the absence of antibodies, through more ancient pathways of complement activation.


Immunology_Janeway

Immunology_Janeway. The complement system is composed of more than 30 different plasma proteins, which are produced mainly by the liver. In the absence of infection, these proteins circulate in an inactive form. In the presence of pathogens or of antibody bound to pathogens, the complement system becomes ‘activated.’ Particular complement proteins interact with each other to form several different pathways of complement activation, all of which have the final outcome of killing the pathogen, either directly or by facilitating its phagocytosis, and inducing inflammatory responses that help to fight infection. There are three pathways of complement activation. As the antibody-triggered pathway of complement activation was discovered first, this became known as the classical pathway of complement activation. The next to be discovered was called the alternative pathway, which can be activated by the presence of the pathogen alone; and the most recently discovered is the lectin pathway, which is activated by


Immunology_Janeway

Immunology_Janeway. next to be discovered was called the alternative pathway, which can be activated by the presence of the pathogen alone; and the most recently discovered is the lectin pathway, which is activated by lectin-type proteins that recognize and bind to carbohydrates on pathogen surfaces.


Immunology_Janeway

Immunology_Janeway. We learned in Section 2-4 that proteolysis can be used as a means of activating antimicrobial proteins. In the complement system, activation by proteolysis is inherent, with many of the complement proteins being proteases that successively cleave and activate one another. The proteases of the complement system are synthesized as inactive pro-enzymes, or zymogens, which become enzymatically active only after proteolytic cleavage, usually by another complement protein. The complement pathways are triggered by proteins that act as pattern recognition receptors to detect the presence of pathogens. This detection activates an initial zymogen, triggering a cascade of proteolysis in which complement zymogens are activated sequentially, each becoming an active protease that cleaves and activates many molecules of the next zymogen in the pathway, amplifying the signal as the cascade proceeds. This results in activation of three distinct effector pathways—inflammation, phagocytosis, and


Immunology_Janeway

Immunology_Janeway. many molecules of the next zymogen in the pathway, amplifying the signal as the cascade proceeds. This results in activation of three distinct effector pathways—inflammation, phagocytosis, and membrane attack—that help eliminate the pathogen. In this way, the detection of even a small number of pathogens produces a rapid response that is greatly amplified at each step. This overall scheme for complement is shown in Fig. 2.13.


Immunology_Janeway

Immunology_Janeway. Fig. 2.13 The complement system proceeds in distinct phases in the elimination of microbes. Proteins that can distinguish self from microbial surfaces (yellow box) activate a proteolytic amplification cascade that ends in the formation of the critical enzymatic activity (green box) of C3 convertase, a family of proteases. This activity is the gateway to three effector arms of complement that produce inflammation (purple), enhance phagocytosis of the microbe (blue), and lyse microbial membranes (pink). We will use this color scheme in the figures throughout this chapter to illustrate which activity each complement protein serves. MOVIE 2.1


Immunology_Janeway

Immunology_Janeway. Nomenclature for complement proteins can seem confusing, so we will start by explaining their names. The first proteins discovered belong to the classical pathway, and they are designated by the letter C followed by a number. The native complement proteins—such as the inactive zymogens—have a simple number designation, for example, C1 and C2. Unfortunately, they were named in the order of their discovery rather than the sequence of reactions. The reaction sequence in the classical pathway, for example, is C1, C4, C2, C3, C5, C6, C7, C8, and C9 (note that not all of these are proteases). Products of cleavage reactions are designated by adding a lowercase letter as a suffix. For example, cleavage of C3 produces a small protein fragment called C3a and the remaining larger fragment, C3b. By convention, the larger fragment for other factors is designated by the suffix b, with one exception. For C2, the larger fragment was named C2a by its discoverers, and this system has been maintained in


Immunology_Janeway

Immunology_Janeway. the larger fragment for other factors is designated by the suffix b, with one exception. For C2, the larger fragment was named C2a by its discoverers, and this system has been maintained in the literature, so we preserve it here. Another exception is the naming of C1q, C1r, and C1s: these are not cleavage products of C1 but are distinct proteins that together comprise C1. The proteins of the alternative pathway were discovered later and are designated by different capital letters, for example, factor B and factor D. Their cleavage products are also designated by the addition of lowercase a and b: thus, the large fragment of B is called Bb and the small fragment Ba. Activated complement components are sometimes designated by a horizontal line, for example, C2a; however, we will not use this convention. All the components of the complement system are listed in Fig. 2.14.


Immunology_Janeway

Immunology_Janeway. Besides acting in innate immunity, complement also influences adaptive immunity. Opsonization of pathogens by complement facilitates their uptake by phagocytic antigen-presenting cells that express complement receptors; this enhances the presentation of pathogen antigens to T cells, which we discuss in more detail in Chapter 6. B cells express receptors for complement proteins that enhance their responses to complement-coated antigens, as we describe later in Chapter 10. In addition, several of the complement fragments can act to influence cytokine production by antigen-presenting cells, thereby influencing the direction and extent of the subsequent adaptive immune response, as we describe in Chapter 11. 2-5 The complement system recognizes features of microbial with C3b.


Immunology_Janeway

Immunology_Janeway. Fig. 2.15 gives a highly simplified preview of the initiation mechanisms and outcomes of complement activation. The three pathways of complement activation are initiated in different ways. The lectin pathway is initiated by soluble carbohydrate-binding proteins—mannose-binding lectin (MBL) and the ficolins—that bind to particular carbohydrate structures on microbial surfaces. Specific proteases, called MBL-associated serine proteases (MASPs), that associate with these recognition proteins then trigger the cleavage of complement proteins and activation of the pathway. The classical pathway is initiated when the complement component C1, which comprises a recognition protein (C1q) associated with proteases (C1r and C1s), either recognizes a microbial surface directly or binds to antibodies already bound to a pathogen. Finally, the alternative pathway can be initiated by spontaneous hydrolysis and activation of the complement component C3, which can then bind directly to microbial


Immunology_Janeway

Immunology_Janeway. already bound to a pathogen. Finally, the alternative pathway can be initiated by spontaneous hydrolysis and activation of the complement component C3, which can then bind directly to microbial surfaces.


Immunology_Janeway

Immunology_Janeway. These three pathways converge at the central and most important step in complement activation. When any of the pathways interacts with a pathogen surface, the enzymatic activity of a C3 convertase is generated. There are various types of C3 convertase, depending on the complement pathway activated, but each is a multisubunit protein with protease activity that cleaves complement component 3 (C3). The C3 convertase is bound covalently to the pathogen surface, where it cleaves C3 to generate large amounts of C3b, the main effector molecule of the complement system; and C3a, a small peptide that binds to The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. The complement system and innate immunity. C3a and C5a recruit phagocytic cells to the site of infection and promote infammation C3a infected tissue Phagocytes with receptors for C3b engulf and destroy the pathogen Completion of the complement cascade leads to formation of a membrane-attack complex (MAC), which disrupts cell membrane and causes cell lysis factor D factor B properdin C3 C1q, C1r, C1s C4 C2 MBL/fcolin, MASP-2 C4 C2 C3a C3 complement receptors All pathways generate a C3 convertase, which cleaves C3, leaving C3b bound to the microbial surface and releasing C3a C3b C6 C5b C8 C7 C9 MAC C3b Phagocyte


Immunology_Janeway

Immunology_Janeway. Fig. 2.15 Complement is a system of soluble pattern recognition receptors and effector molecules that detect and destroy microorganisms. The pathogen-recognition mechanisms of the three complement-activation pathways are shown in the top row, along with the complement components used in the proteolytic cascades leading to formation of a C3 convertase. This enzyme activity cleaves complement component C3 into the small soluble protein C3a and the larger component C3b, which becomes covalently bound to the pathogen surface (middle row). The components are listed by biochemical function in Fig, 2.14 and are described in detail in later figures. The lectin pathway of complement activation (top left) is triggered by the binding of mannose-binding lectin (MBL) or ficolins to carbohydrate residues in microbial cell walls and capsules. The classical pathway (top center) is triggered by binding of C1 either to the pathogen surface or to antibody bound to the pathogen. In the alternative


Immunology_Janeway

Immunology_Janeway. residues in microbial cell walls and capsules. The classical pathway (top center) is triggered by binding of C1 either to the pathogen surface or to antibody bound to the pathogen. In the alternative pathway (top right), soluble C3 undergoes spontaneous hydrolysis in the fluid phase, generating C3(H2O), which is augmented by the action of factors B, D, and P (properdin). All pathways thus converge on the formation of C3b bound to a pathogen and lead to all of the effector activities of complement, which are shown in the bottom row. C3b bound to a pathogen acts as an opsonin, enabling phagocytes that express receptors for C3b to ingest the complement-coated microbe more easily (bottom center). C3b can also bind to C3 convertases to produce another activity, a C5 convertase (detail not shown here), which cleaves C5 to C5a and C5b. C5b triggers the late events of the complement pathway in which the terminal components of complement—C6 to C9—assemble into a membrane-attack complex (MAC)


Immunology_Janeway

Immunology_Janeway. here), which cleaves C5 to C5a and C5b. C5b triggers the late events of the complement pathway in which the terminal components of complement—C6 to C9—assemble into a membrane-attack complex (MAC) that can damage the membrane of certain pathogens (bottom right). C3a and C5a act as chemoattractants that recruit immune-system cells to the site of infection and cause inflammation (bottom left).


Immunology_Janeway

Immunology_Janeway. specific receptors and helps induce inflammation. Cleavage of C3 is the critical step in complement activation and leads directly or indirectly to all the effector activities of the complement system (see Fig. 2.15). C3b binds covalently to the microbial surface and acts as an opsonin, enabling phagocytes that carry receptors for complement to take up and destroy the C3b-coated microbe. Later in the chapter, we will describe the different complement receptors that bind C3b that are involved in this function of complement and how C3b is degraded by a serum protease into inactive smaller fragments called C3f and C3dg. C3b can also bind to the C3 convertases produced by the classical and lectin pathways and form another multisubunit enzyme, the C5 convertase. This cleaves C5, liberating the highly inflammatory peptide C5a and generating C5b. C5b initiates the ‘late’ events of complement activation, in which additional complement proteins interact with C5b to form a membraneattack complex


Immunology_Janeway

Immunology_Janeway. inflammatory peptide C5a and generating C5b. C5b initiates the ‘late’ events of complement activation, in which additional complement proteins interact with C5b to form a membraneattack complex (MAC) on the pathogen surface, creating a pore in the cell membrane that leads to cell lysis (see Fig. 2.15, bottom right).


Immunology_Janeway

Immunology_Janeway. The key feature of C3b is its ability to form a covalent bond with microbial surfaces, which allows the innate recognition of microbes to be translated into effector responses. Covalent bond formation is due to a highly reactive thioester bond that is hidden inside the folded C3 protein and cannot react until C3 is cleaved. When C3 convertase cleaves C3 and releases the C3a fragment, large conformational changes occur in C3b that allow the thioester bond to react with a hydroxyl or amino group on the nearby microbial surface (Fig. 2.16). If no bond is made, the thioester is rapidly hydrolyzed, inactivating C3b, which is one way the alternative pathway is inhibited in healthy individuals. As we will see below, some of the individual components of C3 and C5 convertases differ between the various complement pathways; the components that are different are listed in Fig. 2.17.


Immunology_Janeway

Immunology_Janeway. C3b bound to pathogen surface C3b inactivated by hydrolysis ˜°Before cleavage by C3 convertase, the thioester bond within TED is protected from reacting Cleavage of C3 releases C3a, and a change in conformation of C3b allows the thioester bond to react with a chemical group on the pathogen surface C3b C3aTED TED 23 14 5 6 C3 convertase (C4b2a)pathogen surface thioester bond The reactive thioester group of C3b in TED Gly Glu GlnCys ˜°


Immunology_Janeway

Immunology_Janeway. Fig. 2.16 C3 convertase activates C3 for covalent bonding convertase C4b2a is shown here) and release of C3a from the amino to microbial surfaces by cleaving it into C3a and C3b and terminus of the α chain causes a conformational change in C3b exposing a highly reactive thioester bond in C3b. Top panel: that exposes the thioester bond. This can now react with hydroxyl C3 in blood plasma consists of an α chain and a β chain (formed by or amino groups on molecules on microbial surfaces, covalently proteolytic processing from the native C3 polypeptide) held together bonding C3b to the surface. Bottom right panels: schematic view by a disulfide bond. The thioester-containing domain (TED) of the α of the thioester reaction. If a bond is not made with a microbial chain contains a potentially highly reactive thioester bond (red spot). surface, the thioester is rapidly hydrolyzed (that is, cleaved by water), Bottom left panels: cleavage by C3 convertase (the lectin pathway rendering C3b


Immunology_Janeway

Immunology_Janeway. highly reactive thioester bond (red spot). surface, the thioester is rapidly hydrolyzed (that is, cleaved by water), Bottom left panels: cleavage by C3 convertase (the lectin pathway rendering C3b inactive.


Immunology_Janeway

Immunology_Janeway. The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. Pathways leading to such potent inflammatory and destructive effects—and which have a series of built-in amplification steps—are potentially dangerous and must be tightly regulated. One important safeguard is that the key activated complement components are rapidly inactivated unless they bind to the pathogen surface on which their activation was initiated. There are also several points in the pathway at which regulatory proteins act to prevent the activation of complement on the surfaces of healthy host cells, thereby protecting them from accidental damage, as we shall see later in the chapter. Complement can, however, be activated by dying cells, such as those at sites of ischemic injury, and by cells undergoing apoptosis, or programmed cell death. In these cases, the complement coating helps phagocytes dispose of the dead and dying cells neatly, thus limiting the risk of cell contents being released and triggering an autoimmune response (discussed in Chapter 15).


Immunology_Janeway

Immunology_Janeway. Having introduced some of the main complement components, we are ready for a more detailed account of the three pathways. To help indicate the types of functions carried out by each of the complement components in the tables throughout the rest of the chapter, we will use the color code introduced in Fig. 2.13 and Fig. 2.14: yellow for recognition and activation, green for amplification, purple for inflammation, blue for phagocytosis, and pink for membrane attack. 2-6 The lectin pathway uses soluble receptors that recognize microbial surfaces to activate the complement cascade.


Immunology_Janeway

Immunology_Janeway. Microorganisms typically bear on their surface repeating patterns of molecular structures, known generally as pathogen-associated molecular patterns (PAMPs). The cell walls of Gram-positive and Gram-negative bacteria, for example, are composed of a matrix of proteins, carbohydrates, and lipids in a repetitive array (see Fig. 2.9). The lipoteichoic acids of Gram-positive bacterial cell walls and the lipopolysaccharide of the outer membrane of Gram-negative bacteria are not present on animal cells and are important in the recognition of bacteria by the innate immune system. Similarly, the glycans of yeast surface proteins commonly terminate in mannose residues rather than the sialic acid residues (N-acetylneuraminic acid) that terminate the glycans of vertebrate cells (Fig. 2.18). The lectin pathway uses these features of microbial surfaces to detect and respond to pathogens.


Immunology_Janeway

Immunology_Janeway. Fig. 2.18 The carbohydrate side chains on yeast and vertebrate glycoproteins are terminated with different patterns of sugars. N-linked glycosylation in fungi and animals is initiated by the addition of the same precursor oligosaccharide, Glc3Man9-GlcNAc2 (left panel), to an asparagine residue. In many yeasts this is processed to high-mannose glycans (middle panel). In contrast, in vertebrates, the initial glycan is trimmed and processed, and the N-linked glycoproteins of vertebrates have terminal sialic acid residues (right panel).


Immunology_Janeway

Immunology_Janeway. The lectin pathway can be triggered by any of four different pattern recognition receptors that circulate in blood and extracellular fluids and recognize carbohydrates on microbial surfaces. The first such receptor to be discovered was mannose-binding lectin (MBL), which is shown in Fig. 2.19, and which is synthesized in the liver. MBL is an oligomeric protein built up from a monomer that contains an amino-terminal collagen-like domain and a carboxy-terminal C-type lectin domain (see Section 2-4). Proteins of this type are called collectins. MBL monomers assemble into trimers through the formation of a triple helix by their collagen-like domains. Trimers then assemble into oligomers by disulfide bonding between the cysteine-rich collagen domains. The MBL present in the blood is composed of two to six trimers, with the major forms of human MBL being trimers and tetramers. A single carbohydrate-recognition domain of MBL has a low affinity for mannose, fucose, and N-acetylglucosamine


Immunology_Janeway

Immunology_Janeway. of two to six trimers, with the major forms of human MBL being trimers and tetramers. A single carbohydrate-recognition domain of MBL has a low affinity for mannose, fucose, and N-acetylglucosamine (GlcNAc) residues, which are common on microbial glycans, but does not bind sialic acid residues, which terminate vertebrate glycans. Thus, multimeric MBL has high total binding strength, or avidity, for repetitive carbohydrate structures on a wide variety of microbial surfaces, including Gram-positive and Gram-negative bacteria, mycobacteria, yeasts, and some viruses and parasites, while not interacting with host cells. MBL is present at low concentrations in the plasma of most individuals, but in the presence of infection, its production is increased during the acute-phase response. This is part of the induced phase of the innate immune response and is discussed in Chapter 3.


Immunology_Janeway

Immunology_Janeway. The other three pathogen-recognition molecules used by the lectin pathway are known as ficolins. Although related in overall shape and function to MBL, they have a fibrinogen-like domain, rather than a lectin domain, attached to


Immunology_Janeway

Immunology_Janeway. Mannose-binding lectin (MBL) (left panels) is an oligomeric protein in which two to six clusters of carbohydrate-binding heads arise from a central stalk formed from the collagen-like tails of the MBL monomers. An MBL monomer is composed of a collagen region (red), an α-helical neck region (blue), and a carbohydrate-recognition domain (yellow). Three MBL monomers associate to form a trimer, and between two and six trimers assemble to form a mature MBL molecule (bottom left panel). An MBL molecule associates with MBL-associated serine proteases (MASPs). MBL binds to bacterial surfaces that display a particular spatial arrangement of mannose or fucose residues. The ficolins (right panels) resemble MBL in their overall structure, are associated with MASP-1 and MASP-2, and can activate C4 and C2 after binding to carbohydrate molecules present on microbial surfaces. The carbohydrate-binding domain of ficolins is a fibrinogenlike domain, rather than the lectin domain present in MBL.


Immunology_Janeway

Immunology_Janeway. Fig. 2.19 Mannose-binding lectin and ficolins form complexes with serine proteases and recognize particular carbohydrates on microbial surfaces. collagen helices ˜-helical coiled coils carbohydrate-recognition domains MBL monomers form trimeric clusters of carbohydrate-recognition domains collagen helices ˜-helical coiled coils ÿbronectin domains Ficolins are similar in structure to MBL but have a different carbohydrate-binding domain The complement system and innate immunity. the collagen-like stalk (see Fig. 2.19). The fibrinogen-like domain gives ficolins a general specificity for oligosaccharides containing acetylated sugars, but it does not bind mannose-containing carbohydrates. Humans have three ficolins: L-ficolin (ficolin-2), M-ficolin (ficolin-1), and H-ficolin (ficolin-3). L- and H-ficolin are synthesized by the liver and circulate in the blood; M-ficolin is synthesized and secreted by lung and blood cells.


Immunology_Janeway

Immunology_Janeway. MBL in plasma forms complexes with the MBL-associated serine proteases MASP-1, MASP-2, and MASP-3, which bind MBL as inactive zymogens. When MBL binds to a pathogen surface, a conformational change occurs in MASP-1 that enables it to cleave and activate a MASP-2 molecule in the same MBL complex. Activated MASP-2 can then cleave complement components C4 and C2 (Fig. 2.20). Like MBL, ficolins form oligomers that make a complex with MASP-1 and MASP-2, which similarly activate complement upon recognition of a microbial surface by the ficolin. C4, like C3, contains a buried thioester bond. When MASP-2 cleaves C4, it releases C4a, allowing a conformational change in C4b that exposes the reactive thioester as described for C3b (see Fig. 2.16). C4b bonds covalently via this thioester to the microbial surface nearby, where it then binds one molecule of C2 (see Fig. 2.20). C2 is cleaved by MASP-2, producing C2a, an active serine protease that remains bound to C4b to form C4b2a, which is the C3


Immunology_Janeway

Immunology_Janeway. surface nearby, where it then binds one molecule of C2 (see Fig. 2.20). C2 is cleaved by MASP-2, producing C2a, an active serine protease that remains bound to C4b to form C4b2a, which is the C3 convertase of the lectin pathway. (Remember, C2a is the exception in complement nomenclature.) C4b2a now cleaves many molecules of C3 into C3a and C3b. The C3b fragments bond covalently to the nearby pathogen surface, and the released C3a initiates a local inflammatory response. The complement-activation pathway initiated by ficolins proceeds like the MBL lectin pathway (see Fig. 2.20).


Immunology_Janeway

Immunology_Janeway. Individuals deficient in MBL or MASP-2 experience substantially more respiratory infections by common extracellular bacteria during early childhood, indicating the importance of the lectin pathway for host defense. This susceptibility illustrates the particular importance of innate defense mechanisms in early childhood, when adaptive immune responses are not yet fully developed


Immunology_Janeway

Immunology_Janeway. Binding of mannose-binding lectin or ficolins to their carbohydrate cleaves many molecules of C3 to produce C3b, which binds to the ligands on microbial surfaces induces MASP-1 to cleave and activate pathogen surface, and C3a, an inflammatory mediator. The covalent the serine protease MASP-2. MASP-2 then cleaves C4, exposing attachment of C3b and C4b to the pathogen surface is important in the thioester bond in C4b that allows it to react covalently with the confining subsequent complement activity to pathogen surfaces. pathogen surface. C4b then binds C2, making C2 susceptible to Fig. 2.21 The first protein in the classical pathway of complement activation is C1, which is a complex of C1q, C1r, and C1s. As shown in the micrograph and drawing, C1q is composed of six identical subunits with globular heads (yellow) and long collagen-like tails (red); it has been described as looking like “a bunch of tulips.” The tails combine to bind to two molecules each of C1r and C1s, forming the C1


Immunology_Janeway

Immunology_Janeway. globular heads (yellow) and long collagen-like tails (red); it has been described as looking like “a bunch of tulips.” The tails combine to bind to two molecules each of C1r and C1s, forming the C1 complex C1q:C1r2:C1s2. The heads can bind to the constant regions of immunoglobulin molecules or directly to the pathogen surface, causing a conformational change in C1r, which then cleaves and activates the C1s zymogen (proenzyme). The C1 complex is similar in overall structure to the MBL–MASP complex, and it has an identical function, cleaving C4 and C2 to form the C3 convertase C4b2a (see Fig. 2.20). Photograph (×500,000) courtesy of K.B.M. Reid.


Immunology_Janeway

Immunology_Janeway. but the maternal antibodies transferred across the placenta and present in the mother’s milk are gone. Other members of the collectin family are the surfactant proteins A and D (SP-A and SP-D), which are present in the fluid that bathes the epithelial surfaces of the lung. There they coat the surfaces of pathogens, making them more susceptible to phagocytosis by macrophages that have left the subepithelial tissues to enter the alveoli. Because SP-A and SP-D do not associate with MASPs, they do not activate complement.


Immunology_Janeway

Immunology_Janeway. We have used MBL here as our prototype activator of the lectin pathway, but the ficolins are more abundant than MBL in plasma and so may be more important in practice. L-ficolin recognizes acetylated sugars such as GlcNAc and N-acetylgalactosamine (GalNAc), and particularly recognizes lipoteichoic acid, a component of the cell walls of Gram-positive bacteria that contains GalNAc. It can also activate complement after binding to a variety of capsulated bacteria. M-ficolin also recognizes acetylated sugar residues; H-ficolin shows a more restricted binding specificity, for d-fucose and galactose, and has only been linked to activity against the Gram-positive bacterium Aerococcus viridans, a cause of bacterial endocarditis. 2-7 The classical pathway is initiated by activation of the C1 complex and is homologous to the lectin pathway.


Immunology_Janeway

Immunology_Janeway. 2-7 The classical pathway is initiated by activation of the C1 complex and is homologous to the lectin pathway. In its overall scheme, the classical pathway is similar to the lectin pathway, except that it uses a pathogen sensor known as the C1 complex, or C1. Because C1 interacts directly with some pathogens but can also interact with antibodies, C1 allows the classical pathway to function both in innate immunity, which we describe now, and in adaptive immunity, which we examine in more detail in Chapter 10.


Immunology_Janeway

Immunology_Janeway. Like the MBL–MASP complex, the C1 complex is composed of a large subunit (C1q), which acts as the pathogen sensor, and two serine proteases (C1r and C1s), which are initially in their inactive form (Fig. 2.21). C1q is a hexamer of trimers, composed of monomers that contain an amino-terminal globular domain and a carboxy-terminal collagen-like domain. The trimers assemble through interactions of the collagen-like domains, bringing the globular domains together to form a globular head. Six of these trimers assemble to form a complete C1q molecule, which has six globular heads held together by their collagen-like tails. C1r and C1s are closely related to MASP-2, and somewhat more distantly related to MASP-1 and MASP-3; all five enzymes are likely to have evolved from the duplication of a gene for a common precursor. C1r and C1s interact noncovalently and form tetramers that fold into the arms of C1q, with at least part of the C1r:C1s complex being external to C1q.


Immunology_Janeway

Immunology_Janeway. The recognition function of C1 resides in the six globular heads of C1q. When two or more of these heads interact with a ligand, this causes a conformational change in the C1r:C1s complex, which leads to the activation of an autocatalytic enzymatic activity in C1r; the active form of C1r then cleaves its associated C1s to generate an active serine protease. The activated C1s acts on the next two components of the classical pathway, C4 and C2. C1s cleaves C4 to produce C4b, which binds covalently to the pathogen surface as described earlier for the lectin pathway (see Fig. 2.20). C4b then also binds one molecule of C2, which is cleaved by C1s to produce the serine protease C2a. This produces the active C3 convertase C4b2a, which is the C3 convertase of both the lectin and the classical pathways. However, because it was first discovered as part of the classical pathway, C4b2a is often known as the classical C3 convertase (see Fig. 2.17). The proteins involved in the classical pathway,


Immunology_Janeway

Immunology_Janeway. However, because it was first discovered as part of the classical pathway, C4b2a is often known as the classical C3 convertase (see Fig. 2.17). The proteins involved in the classical pathway, and their active forms, are listed in Fig. 2.22.


Immunology_Janeway

Immunology_Janeway. C1q can attach itself to the surface of pathogens in several different ways. One is by binding directly to surface components on some bacteria, including The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. certain proteins of bacterial cell walls and polyanionic structures such as the lipoteichoic acid on Gram-positive bacteria. A second is through binding to C-reactive protein, an acute-phase protein in human plasma that binds to phosphocholine residues in bacterial surface molecules such as pneumococcal C polysaccharide—hence the name C-reactive protein. We discuss the acute-phase proteins in detail in Chapter 3. However, a main function of C1q in an immune response is to bind to the constant, or Fc, regions of antibodies (see Section 1-9) that have bound pathogens via their antigen-binding sites. C1q thus links the effector functions of complement to recognition provided by adaptive immunity. This might seem to limit the usefulness of C1q in fighting the first stages of an infection, before the adaptive immune response has generated pathogen-specific antibodies. However, some antibodies, called natural antibodies, are produced by the immune system in the apparent absence of


Immunology_Janeway

Immunology_Janeway. before the adaptive immune response has generated pathogen-specific antibodies. However, some antibodies, called natural antibodies, are produced by the immune system in the apparent absence of infection. These antibodies have a low affinity for many microbial pathogens and are highly cross-reactive, recognizing common membrane constituents such as phosphocholine and even recognizing some antigens of the body’s own cells (that is, self antigens). Natural antibodies may be produced in response to commensal microbiota or to self antigens, but do not seem to be the consequence of an adaptive immune response to infection by pathogens. Most natural antibody is of the isotype, or class, known as IgM (see Sections 1-9 and 1-20) and represents a considerable amount of the total IgM circulating in humans. IgM is the class of antibody most efficient at binding C1q, making natural antibodies an effective means of activating complement on microbial surfaces immediately after infection and leading


Immunology_Janeway

Immunology_Janeway. humans. IgM is the class of antibody most efficient at binding C1q, making natural antibodies an effective means of activating complement on microbial surfaces immediately after infection and leading to the clearance of bacteria such as Streptococcus pneumoniae (the pneumococcus) before they become dangerous.


Immunology_Janeway

Immunology_Janeway. 2-8 Complement activation is largely confined to the surface on which it is initiated. We have seen that both the lectin and the classical pathways of complement activation are initiated by proteins that bind to pathogen surfaces. During the triggered enzyme cascade that follows, it is important that activating events are confined to this same site, so that C3 activation also occurs on the surface of the Fig. 2.23 The alternative pathway of complement activation can amplify the classical or the lectin pathway by forming an alternative C3 convertase and depositing more C3b molecules on the pathogen. C3b deposited by the classical or lectin pathway can bind factor B, making it susceptible to cleavage by factor D. The C3bBb complex is the C3 convertase of the alternative pathway of complement activation, and its action, like that of C4b2a, results in the deposition of many molecules of C3b on the pathogen surface.


Immunology_Janeway

Immunology_Janeway. pathogen and not in the plasma or on host-cell surfaces. This is achieved principally by the covalent binding of C4b to the pathogen surface. In innate immunity, C4 cleavage is catalyzed by a ficolin or MBL complex that is bound to the pathogen surface, and so the C4b cleavage product can bind adjacent proteins or carbohydrates on the pathogen surface. If C4b does not rapidly form this bond, the thioester bond is cleaved by reaction with water and C4b is irreversibly inactivated. This helps to prevent C4b from diffusing from its site of activation on the microbial surface and becoming attached to healthy host cells.


Immunology_Janeway

Immunology_Janeway. C2 becomes susceptible to cleavage by C1s only when it is bound by C4b, and the active C2a serine protease is thereby also confined to the pathogen surface, where it remains associated with C4b, forming the C3 convertase C4b2a. Cleavage of C3 to C3a and C3b is thus also confined to the surface of the pathogen. Like C4b, C3b is inactivated by hydrolysis unless its exposed thioester rapidly makes a covalent bond (see Fig. 2.16), and it therefore opsonizes only the surface on which complement activation has taken place. Opsonization by C3b is more effective when antibodies are also bound to the pathogen surface, as phagocytes have receptors for both complement and Fc receptors that bind the Fc region of antibody (see Sections 1-20 and 10-20). Because the reactive forms of C3b and C4b are able to form a covalent bond with any adjacent protein or carbohydrate, when complement is activated by bound antibody, a proportion of the reactive C3b or C4b will become linked to the antibody


Immunology_Janeway

Immunology_Janeway. are able to form a covalent bond with any adjacent protein or carbohydrate, when complement is activated by bound antibody, a proportion of the reactive C3b or C4b will become linked to the antibody molecules themselves. Antibody that is chemically cross-linked to complement is likely the most efficient trigger for phagocytosis.


Immunology_Janeway

Immunology_Janeway. 2-9 The alternative pathway is an amplification loop for C3b formation that is accelerated by properdin in the presence of pathogens.


Immunology_Janeway

Immunology_Janeway. Although probably the most ancient of the complement pathways, the alternative pathway is so named because it was discovered as a second, or ‘alternative,’ pathway for complement activation after the classical pathway had been defined. Its key feature is its ability to be spontaneously activated. It has a unique C3 convertase, the alternative pathway C3 convertase, that differs from the C4b2a convertase of the lectin or classical pathways (see Fig. 2.17). The alternative pathway C3 convertase is composed of C3b itself bound to Bb, which is a cleavage fragment of the plasma protein factor B. This C3 convertase, designated C3bBb, has a special place in complement activation because, by producing C3b, it can generate more of itself. This means that once some C3b has been formed, by whichever pathway, the alternative pathway can act as an amplification loop to increase C3b production rapidly.


Immunology_Janeway

Immunology_Janeway. The alternative pathway can be activated in two different ways. The first is by the action of the lectin or classical pathway. C3b generated by either of these pathways and covalently linked to a microbial surface can bind factor B (Fig. 2.23). This alters the conformation of factor B, enabling a plasma protease The complement system and innate immunity. The C3(H2O)Bb complex is a C3 convertase, cleaving more C3 into C3a and C3b. C3b is rapidly inactivated unless it binds to a cell surface


Immunology_Janeway

Immunology_Janeway. The complement system and innate immunity. The C3(H2O)Bb complex is a C3 convertase, cleaving more C3 into C3a and C3b. C3b is rapidly inactivated unless it binds to a cell surface Complement component C3 hydrolyzes spontaneously in plasma to give C3(H2O), which binds factor B and enables the bound factor B to be cleaved by factor D (first panel). The resulting ‘soluble C3 convertase’ cleaves C3 to give C3a and C3b, which can attach to host cells or pathogen surfaces (second panel). Covalently bound to the cell surface, C3b binds factor B; in turn, factor B is rapidly cleaved by factor D to Bb, which remains bound to C3b to form a C3 convertase (C3bBb), and Ba, which is released (third panel). This convertase functions in the alternative pathway as the C4b2a C3 convertase does in the lectin and classical pathways (see Fig. 2.17).


Immunology_Janeway

Immunology_Janeway. called factor D to cleave it into Ba and Bb. Bb remains stably associated with C3b, forming the C3bBb C3 convertase. The second way of activating the alternative pathway involves the spontaneous hydrolysis (known as ‘tickover’) of the thioester bond in C3 to form C3(H2O), as shown in Fig. 2.24. C3 is abundant in plasma, and tickover causes a steady, low-level production of C3(H2O). This C3(H2O) can bind factor B, which is then cleaved by factor D, producing a short-lived fluid-phase C3 convertase, C3(H2O)Bb. Although formed in only small amounts by C3 tickover, fluid-phase C3(H2O)Bb can cleave many molecules of C3 to C3a and C3b. Much of this C3b is inactivated by hydrolysis, but some attaches covalently via its thioester bond to the surfaces of any microbes present. C3b formed in this way is no different from C3b produced by the lectin or classical pathways and binds factor B, leading to the formation of C3 convertase and a stepping up of C3b production (see Fig. 2.23).


Immunology_Janeway

Immunology_Janeway. On their own, the alternative pathway C3 convertases C3bBb and C3(H2O) Bb are very short-lived. They are, however, stabilized by binding the plasma protein properdin (factor P) (Fig. 2.25). Properdin is made by neutrophils and stored in secondary granules. It is released when neutrophils are activated by the presence of pathogens. Properdin may have some properties of a pattern recognition receptor, since it can bind to some microbial surfaces. Properdindeficient patients are particularly susceptible to infections with Neisseria meningitidis, the main agent of bacterial meningitis. Properdin’s ability to bind to bacterial surfaces may direct the activity of the alternative complement pathway to these pathogens, thus aiding their removal by phagocytosis. Properdin can also bind to mammalian cells that are undergoing apoptosis or have been damaged or modified by ischemia, viral infection, or antibody binding, leading to the deposition of C3b on these cells and facilitating their removal


Immunology_Janeway

Immunology_Janeway. cells that are undergoing apoptosis or have been damaged or modified by ischemia, viral infection, or antibody binding, leading to the deposition of C3b on these cells and facilitating their removal by phagocytosis. The distinctive components of the alternative pathway are listed in Fig. 2.26.


Immunology_Janeway

Immunology_Janeway. Fig. 2.25 Properdin stabilizes the alternative pathway C3 convertase on pathogen surfaces. Bacterial surfaces do not express complement-regulatory proteins and favor the binding of properdin (factor P), which stabilizes the C3bBb convertase. This convertase activity is the equivalent of C4b2a of the classical pathway. C3bBb then cleaves many more molecules of C3, coating the pathogen surface with bound C3b. C3bBb complex is a C3 convertase and deposits many molecules of C3b on the pathogen surface Opsonization, activation of terminal complement components


Immunology_Janeway

Immunology_Janeway. C3bBb complex is a C3 convertase and deposits many molecules of C3b on the pathogen surface Opsonization, activation of terminal complement components Fig. 2.27 Complement activation spares host cells, which are protected by complement-regulatory proteins. If C3bBb forms on the surface of host cells, it is rapidly inactivated by complement-regulatory proteins expressed by the host cell: complement receptor 1 (CR1), decay-accelerating factor (DAF), and membrane cofactor of proteolysis (MCP). Host-cell surfaces also favor the binding of factor H from plasma. CR1, DAF, and factor H displace Bb from C3b, and CR1, MCP, and factor H catalyze the cleavage of bound C3b by the plasma protease factor I to produce inactive C3b (known as iC3b). 2-10 Membrane and plasma proteins that regulate the formation and stability of C3 convertases determine the extent of complement activation.


Immunology_Janeway

Immunology_Janeway. 2-10 Membrane and plasma proteins that regulate the formation and stability of C3 convertases determine the extent of complement activation. Several mechanisms ensure that complement activation will proceed only on the surface of a pathogen or on damaged host cells, and not on normal host cells and tissues. After initial complement activation by any pathway, the extent of amplification via the alternative pathway is critically dependent on the stability of the C3 convertase C3bBb. This stability is controlled by both positive and negative regulatory proteins. We have already described how properdin acts as a positive regulatory protein on foreign surfaces, such as those of bacteria or damaged host cells, by stabilizing C3bBb.


Immunology_Janeway

Immunology_Janeway. Several negative regulatory proteins, present in plasma and in host-cell membranes, protect healthy host cells from the injurious effects of inappropriate complement activation on their surfaces. These complement-regulatory proteins interact with C3b and either prevent the convertase from forming or promote its rapid dissociation (Fig. 2.27). For example, a membrane-attached protein known as decay-accelerating factor (DAF or CD55) competes with factor B for binding to C3b on the cell surface and can displace Bb from a convertase that has already formed. Convertase formation can also be prevented by cleaving C3b to an inactive derivative, iC3b. This is achieved by a plasma protease, factor I, in conjunction with C3b-binding proteins that act as cofactors, such as membrane cofactor of proteolysis (MCP or CD46), another host-cell membrane protein (see Fig. 2.27). Cell-surface complement receptor type 1 (CR1, also known as CD35) behaves similarly to DAF and MCP in that it inhibits C3


Immunology_Janeway

Immunology_Janeway. (MCP or CD46), another host-cell membrane protein (see Fig. 2.27). Cell-surface complement receptor type 1 (CR1, also known as CD35) behaves similarly to DAF and MCP in that it inhibits C3 convertase formation and promotes the catabolism of C3b to inactive products, but it has a more limited tissue distribution. Factor H is another complement-regulatory protein in plasma that binds C3b, and like CR1, it is able to compete with factor B to displace Bb from the convertase; in addition, it acts as a cofactor for factor I. Factor H binds preferentially to C3b bound to vertebrate cells because it has an affinity for the sialic acid residues present on their cell surfaces (see Fig. 2.18). Thus, the amplification loop of the alternative pathway is allowed to proceed on the surface of a pathogen or on damaged host cells, but not on normal host cells or on tissues that express these negative regulatory proteins.


Immunology_Janeway

Immunology_Janeway. The C3 convertase of the classical and lectin pathways (C4b2a) is molecularly distinct from that of the alternative pathway (C3bBb). However, understanding of the complement system is simplified somewhat by recognition of the close evolutionary relationships between the different complement proteins (Fig. 2.28). Thus the complement zymogens, factor B and C2, are closely related proteins encoded by homologous genes located in tandem within the major The complement system and innate immunity. histocompatibility complex (MHC) on human chromosome 6. Furthermore, their respective binding partners, C3 and C4, both contain thioester bonds that provide the means of covalently attaching the C3 convertases to a pathogen surface.


Immunology_Janeway

Immunology_Janeway. Only one component of the alternative pathway seems entirely unrelated to its functional equivalents in the classical and lectin pathways: the initiating serine protease, factor D. Factor D can also be singled out as the only activating protease of the complement system to circulate as an active enzyme rather than a zymogen. This is both necessary for the initiation of the alternative pathway (through the cleavage of factor B bound to spontaneously activated C3) and safe for the host, because factor D has no other substrate than factor B bound to C3b. This means that factor D finds its substrate only at pathogen surfaces and at a very low level in plasma, where the alternative pathway of complement activation can be allowed to proceed. 2-11 Complement developed early in the evolution of multicellular organisms.


Immunology_Janeway

Immunology_Janeway. The complement system was originally known only from vertebrates, but homologs of C3 and factor B and a prototypical ‘alternative pathway’ have been discovered in nonchordate invertebrates. This is not altogether surprising since C3, which is cleaved and activated by serine proteases, is evolutionarily related to the serine protease inhibitor α2-macroglobulin, whose first appearance likely was in an ancestor to all modern vertebrates. The amplification loop of the alternative pathway also has an ancestral origin, as it is present in echinoderms (which include sea urchins and sea stars) and is based on a C3 convertase formed by the echinoderm homologs of C3 and factor B. These factors are expressed by phagocytic cells called amoeboid coelomocytes present in the coelomic fluid. Expression of C3 by these cells increases when bacteria are present. This simple system seems to function to opsonize bacterial cells and other foreign particles and facilitate their uptake by coelomocytes. C3


Immunology_Janeway

Immunology_Janeway. of C3 by these cells increases when bacteria are present. This simple system seems to function to opsonize bacterial cells and other foreign particles and facilitate their uptake by coelomocytes. C3 homologs in invertebrates are clearly related to each other. They all contain the distinctive thioester linkage and form a family of proteins, the thioester proteins, or TEPs. In the mosquito Anopheles, the production of protein TEP1 is induced in response to infection, and the protein may directly bind to bacterial surfaces to mediate phagocytosis of Gram-negative bacteria. Some form of C3 activity may even predate the evolution of the Bilateria—animals with bilateral symmetry, flatworms being


Immunology_Janeway

Immunology_Janeway. Fig. 2.28 There is a close evolutionary relationship among the factors of the alternative, lectin, and classical pathways of complement activation.


Immunology_Janeway

Immunology_Janeway. Most of the factors are either identical or the homologous products of genes that have duplicated and then diverged in sequence. The proteins C4 and C3 are homologous and contain the unstable thioester bond by which their large fragments, C4b and C3b, bind covalently to membranes. The genes encoding proteins C2 and factor B are adjacent in the MHC region of the genome and arose by gene duplication. The regulatory proteins factor H, CR1, and C4BP share a repeat sequence common to many complement-regulatory proteins. The greatest divergence between the pathways is in their initiation: in the classical pathway the C1 complex binds either to certain pathogens or to bound antibody, and in the latter circumstance it serves to convert antibody binding into enzyme activity on a specific surface; in the lectin pathway, mannose-binding lectin (MBL) associates with a serine protease, activating MBL-associated serine protease (MASP), to serve the same function as C1r:C1s; in the alternative


Immunology_Janeway

Immunology_Janeway. in the lectin pathway, mannose-binding lectin (MBL) associates with a serine protease, activating MBL-associated serine protease (MASP), to serve the same function as C1r:C1s; in the alternative pathway this enzyme activity is provided by factor D.


Immunology_Janeway

Immunology_Janeway. Fig. 2.29 Complement component C5 is cleaved when captured by a C3b molecule that is part of a C5 convertase complex. As shown in the top panel, C5 convertases are formed when C3b binds either the classical or lectin pathway C3 convertase C4b2a to form C4b2a3b, or the alternative pathway C3 convertase C3bBb to form C3b2Bb. C5 binds to C3b in these complexes (center panel). The bottom panel shows that C5 is cleaved by the active enzyme C2a or Bb to form C5b and the inflammatory mediator C5a. Unlike C3b and C4b, C5b is not covalently bound to the cell surface. The production of C5b initiates the assembly of the terminal complement components. the most primitive modern representatives—because genomic evidence of C3, factor B, and some later-acting complement components has been found in the Anthozoa (corals and sea anemones).


Immunology_Janeway

Immunology_Janeway. After its initial appearance, the complement system seems to have evolved by the acquisition of new activation pathways that allow specific targeting of microbial surfaces. The first to evolve was likely the ficolin pathway, which is present both in vertebrates and in some closely related invertebrates, such as the urochordates. Evolutionarily, the ficolins may predate the collectins, which are also first seen in the urochordates. Homologs of MBL and of the classical pathway complement component C1q, both collectins, have been identified in the genome of the ascidian urochordate Ciona (sea squirt). Two invertebrate homologs of mammalian MASPs also have been identified in Ciona, and it seems likely that they may be able to cleave and activate C3. Thus, the minimal complement system of the echinoderms appears to have been expanded in the urochordates by the recruitment of a specific activation system that may target C3 deposition onto microbial surfaces. This also suggests that when


Immunology_Janeway

Immunology_Janeway. echinoderms appears to have been expanded in the urochordates by the recruitment of a specific activation system that may target C3 deposition onto microbial surfaces. This also suggests that when adaptive immunity evolved, much later, the ancestral antibody molecule used an already diversified C1q-like collectin member to activate the complement pathway, and that the complement activation system evolved further by use of this collectin and its associated MASPs to become the initiating components of the classical complement pathway, namely, C1q, C1r, and C1s.


Immunology_Janeway

Immunology_Janeway. 2-12 Surface-bound C3 convertase deposits large numbers of convertase activity.


Immunology_Janeway

Immunology_Janeway. We now return to the present-day complement system. The formation of C3 convertases is the point at which the three pathways of complement activation converge. The convertase of the lectin and classical pathways, C4b2a, and the convertase of the alternative pathway, C3bBb, initiate the same subsequent events—they cleave C3 to C3b and C3a. C3b binds covalently through its thioester bond to adjacent molecules on the pathogen surface; otherwise it is inactivated by hydrolysis. C3 is the most abundant complement protein in plasma, occurring at a concentration of 1.2 mg/ml, and up to 1000 molecules of C3b can bind in the vicinity of a single active C3 convertase (see Fig. 2.23). Thus, the main effect of complement activation is to deposit large quantities of C3b on the surface of the infecting pathogen, where the C3b forms a covalently bonded coat that can signal the ultimate destruction of the pathogen by phagocytes.


Immunology_Janeway

Immunology_Janeway. The next step in the complement cascade is the generation of the C5 convertases. C5 is a member of the same family of proteins as C3, C4, α2-macroglobulin, and the thioester-containing proteins (TEPs) of invertebrates. C5 does not form an active thioester bond during its synthesis but, like C3 and C4, it is cleaved by a specific protease into C5a and C5b fragments, each of which exerts specific downstream actions that are important in propagating the complement cascade. In the classical and the lectin pathways, a C5 convertase is formed by the binding of C3b to C4b2a to yield C4b2a3b. The C5 convertase of the alternative pathway is formed by the binding of C3b to the C3bBb convertase to form C3b2Bb. A C5 is captured by these C5 convertase complexes through binding to an acceptor site on C3b, and is thus rendered susceptible to cleavage by the serine protease activity of C2a or Bb. This reaction, which generates C5b and C5a, is much more limited than cleavage of C3, because C5 can be


Immunology_Janeway

Immunology_Janeway. C3b, and is thus rendered susceptible to cleavage by the serine protease activity of C2a or Bb. This reaction, which generates C5b and C5a, is much more limited than cleavage of C3, because C5 can be cleaved only when it binds to C3b that is in turn bound to C4b2a or C3bBb to form the active C5 convertase complex. Thus, complement activated by all three pathways leads to the binding of large numbers of C3b molecules on the surface of the pathogen, the generation of a more limited number of C5b molecules, and the release of C3a and a smaller amount of C5a (Fig. 2.29).


Immunology_Janeway

Immunology_Janeway. The complement system and innate immunity. 2-13 Ingestion of complement-tagged pathogens by phagocytes is mediated by receptors for the bound complement proteins. The most important action of complement is to facilitate the uptake and destruction of pathogens by phagocytic cells. This occurs by the specific recognition of bound complement components by complement receptors (CRs) on phagocytes. These complement receptors bind pathogens opsonized with complement components: opsonization of pathogens is a major function of C3b and its proteolytic derivatives. C4b also acts as an opsonin but has a relatively minor role, largely because so much more C3b than C4b is generated.


Immunology_Janeway

Immunology_Janeway. The known receptors for bound complement components C5a and C3a are listed, with their functions and distributions, in Fig. 2.30. The C3b receptor CR1, described in Section 2-10, is a negative regulator of complement activation (see Fig. 2.27). CR1 is expressed on many types of immune cells, including macrophages and neutrophils. Binding of C3b to CR1 cannot by itself stimulate phagocytosis, but it can lead to phagocytosis in the presence of other immune mediators that activate macrophages. For example, the small complement fragment C5a can activate macrophages to ingest bacteria bound to their CR1 receptors (Fig. 2.31). C5a binds to another receptor expressed by macrophages, the C5a receptor, which has seven membrane-spanning domains. Receptors of this type transduce their signals via intracellular guanine-nucleotide-binding proteins called G proteins and are known generally as G-protein-coupled receptors (GPCRs); they are discussed in Section 3-2. C5L2 (GPR77), expressed by


Immunology_Janeway

Immunology_Janeway. via intracellular guanine-nucleotide-binding proteins called G proteins and are known generally as G-protein-coupled receptors (GPCRs); they are discussed in Section 3-2. C5L2 (GPR77), expressed by neutrophils and macrophages, is a


Immunology_Janeway

Immunology_Janeway. Fig. 2.30 Distribution and function of cell-surface receptors for complement proteins. A variety of complement receptors are specific for bound C3b and its cleavage products (iC3b and C3dg). CR1 and CR3 are important in inducing the phagocytosis of bacteria with complement components bound to their surface. CR2 is found mainly on B cells, where it is part of the B-cell co-receptor complex CR1 and CR2 share structural features with the complement-regulatory proteins that bind C3b and C4b. CR3 and CR4 are integrins composed of integrin β2 paired with either integrin αM (CD11b) or integrin αX (CD11c), respectively (see Appendix II); CR3, also called Mac-1, is also important for leukocyte adhesion and migration, as we shall see in Chapter 3, whereas CR4 is only known to function in phagocytosis. The receptors for C5a and C3a are seven-span G-protein-coupled receptors. FDC, follicular dendritic cells; these are not involved in innate immunity and are discussed in later chapters.


Immunology_Janeway

Immunology_Janeway. Fig. 2.31 The anaphylatoxin C5a can enhance the phagocytosis of microorganisms opsonized in an innate immune response. Activation of complement leads to the deposition of C3b on the surface of microorganisms (left panel). C3b can be bound by the complement receptor CR1 on the surface of phagocytes, but this on its own is insufficient to induce phagocytosis (center panel). Phagocytes also express receptors for the anaphylatoxin C5a, and binding of C5a will now activate the cell to phagocytose microorganisms bound through CR1 (right panel).


Immunology_Janeway

Immunology_Janeway. Fig. 2.32 The cleavage products of C3b are recognized by different complement receptors. After C3b is deposited on the surface of pathogens, it can undergo several conformational changes that alter its interaction with complement receptors. Factor I and MCP can cleave the C3f fragment from C3b, producing iC3b, which is a ligand for the complement receptors CR2, CR3, and CR4, but not CR1. Factor I and CR1 cleave iC3b to release C3c, leaving C3dg bound. C3dg is then recognized by CR2. When only C3b binds to CR1, bacteria are not phagocytosed nonsignaling receptor that acts as a decoy receptor for C5a and may regulate activity of the C5a receptor. Proteins associated with the extracellular matrix, such as fibronectin, can also contribute to phagocyte activation; these are encountered when phagocytes are recruited to connective tissue and activated there.


Immunology_Janeway

Immunology_Janeway. Four other complement receptors—CR2 (also known as CD21), CR3 (CD11b:CD18), CR4 (CD11c:CD18), and CRIg (complement receptor of the immunoglobulin family)—bind to forms of C3b that have been cleaved by factor I but that remain attached to the pathogen surface. Like several other key components of complement, C3b is subject to regulatory mechanisms that cleave it into derivatives, such as iC3b, that cannot form an active convertase. C3b bound to the microbial surface can be cleaved by factor I and MCP to remove the small fragment C3f, leaving the inactive iC3b form bound to the surface (Fig. 2.32). iC3b is recognized by several complement receptors—CR2, CR3, CR4, and CRIg. Unlike the binding of iC3b to CR1, the binding of iC3b to the receptor CR3 is sufficient on its own to stimulate phagocytosis. Factor I and CR1 cleave iC3b to release C3c, leaving C3dg bound to the pathogen. C3dg is recognized only by CR2. CR2 is found on B cells as part of a co-receptor complex that can augment the


Immunology_Janeway

Immunology_Janeway. Factor I and CR1 cleave iC3b to release C3c, leaving C3dg bound to the pathogen. C3dg is recognized only by CR2. CR2 is found on B cells as part of a co-receptor complex that can augment the signal received through the antigen-specific immunoglobulin receptor. Thus, a B cell whose antigen receptor is specific for an antigen of a pathogen will receive a strong signal on binding this antigen if it or the pathogen is also coated with C3dg. The activation of complement can therefore contribute to producing a strong antibody response.


Immunology_Janeway

Immunology_Janeway. The importance of opsonization by C3b and its inactive fragments in destroying extracellular pathogens can be seen in the effects of various complement deficiencies. For example, individuals deficient in C3 or in molecules that catalyze C3b deposition show an increased susceptibility to infection by a wide range of extracellular bacteria, including Streptococcus pneumoniae. We describe the effects of various defects in complement and the diseases they cause in Chapter 13. The complement system and innate immunity. 2-14 The small fragments of some complement proteins initiate a local inflammatory response.


Immunology_Janeway

Immunology_Janeway. The small complement fragments C3a and C5a act on specific receptors on endothelial cells and mast cells (see Fig. 2.30) to produce local inflammatory responses. Like C5a, C3a also signals through a G-protein-coupled receptor, discussed in more detail in Chapter 3. C4a, although generated during C4 cleavage, is not potent at inducing inflammation, is inactive at C3a and C5a receptors, and seems to lack a receptor of its own. When produced in large amounts or injected systemically, C3a and C5a induce a generalized circulatory collapse, producing a shocklike syndrome similar to that seen in a systemic allergic reaction involving antibodies of the IgE class, discussed in Chapter 14. Such a reaction is termed anaphylactic shock, and these small fragments of complement are therefore often referred to as anaphylatoxins. C5a has the highest specific biological activity, but both C3a and C5a induce the contraction of smooth muscle and increase vascular permeability and act on the endothelial


Immunology_Janeway

Immunology_Janeway. to as anaphylatoxins. C5a has the highest specific biological activity, but both C3a and C5a induce the contraction of smooth muscle and increase vascular permeability and act on the endothelial cells lining blood vessels to induce the synthesis of adhesion molecules. In addition, C3a and C5a can activate the mast cells that populate submucosal tissues to release inflammatory molecules such as histamine and the cytokine tumor necrosis factor-α (TNF-α), which cause similar effects. The changes induced by C5a and C3a recruit antibody, complement, and phagocytic cells to the site of an infection (Fig. 2.33), and the increased fluid in the tissues hastens the movement of pathogen-bearing antigen-presenting cells to the local lymph nodes, contributing to the prompt initiation of the adaptive immune response.


Immunology_Janeway

Immunology_Janeway. Small complement-cleavage products act on blood vessels to increase vascular permeability and cell-adhesion molecules Migration of macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes is increased. Microbicidal activity of macrophages and PMNs is also increased


Immunology_Janeway

Immunology_Janeway. Fig. 2.33 Local inflammatory responses can be induced by small complement fragments, especially C5a. The small complement fragments are differentially active: C5a is more active than C3a; C4a is weak or inactive. C5a and C3a cause local inflammatory responses by acting directly on local blood vessels, stimulating an increase in blood flow, increased vascular permeability, and increased binding of phagocytes to endothelial cells. C3a and C5a also activate mast cells (not shown) to release mediators, such as histamine and TNF-α, that contribute to the inflammatory response. The increase in vessel diameter and permeability leads to the accumulation of fluid and protein in the surrounding tissue. Fluid accumulation increases lymphatic drainage, bringing pathogens and their antigenic components to nearby lymph nodes. The antibodies, complement, and cells thus recruited participate in pathogen clearance by enhancing phagocytosis. The small complement fragments can also directly increase the


Immunology_Janeway

Immunology_Janeway. to nearby lymph nodes. The antibodies, complement, and cells thus recruited participate in pathogen clearance by enhancing phagocytosis. The small complement fragments can also directly increase the activity of the phagocytes.


Immunology_Janeway

Immunology_Janeway. Deficiency of the C8 Complement Component C5a also acts directly on neutrophils and monocytes to increase their adherence to vessel walls, their migration toward sites of antigen deposition, and their ability to ingest particles; it also increases the expression of CR1 and CR3 on the surfaces of these cells. In this way, C5a, and to a smaller extent C3a and C4a, act in concert with other complement components to hasten the destruction of pathogens by phagocytes. 2-15 The terminal complement proteins polymerize to form pores in membranes that can kill certain pathogens.


Immunology_Janeway

Immunology_Janeway. 2-15 The terminal complement proteins polymerize to form pores in membranes that can kill certain pathogens. One of the important effects of complement activation is the assembly of the terminal components of complement (Fig. 2.34) to form a membrane-attack complex. The reactions leading to the formation of this complex are shown schematically in Fig. 2.35. The end result is a pore in the lipid bilayer membrane that destroys membrane integrity. This is thought to kill the pathogen by destroying the proton gradient across the pathogen’s cell membrane.


Immunology_Janeway

Immunology_Janeway. The first step in the formation of the membrane-attack complex is the cleavage of C5 by a C5 convertase to release C5b (see Fig. 2.29). In the next stages (see Fig. 2.35), C5b initiates the assembly of the later complement components and their insertion into the cell membrane. The process begins when one molecule of C5b binds one molecule of C6, and the C5b6 complex then binds one molecule of C7. This reaction leads to a conformational change in the constituent molecules, with the exposure of a hydrophobic site on C7, which inserts into the lipid bilayer. Similar hydrophobic sites are exposed on the later components C8 and C9 when they are bound to the complex, allowing these proteins also to insert into the lipid bilayer. C8 is a complex of two proteins, C8β and C8α-γ. The C8β protein binds to C5b, and the binding of C8β to the membrane-associated C5b67 complex allows the hydrophobic domain of C8α-γ to insert into the lipid bilayer. Finally, C8α-γ induces the polymerization of 10–16


Immunology_Janeway

Immunology_Janeway. to C5b, and the binding of C8β to the membrane-associated C5b67 complex allows the hydrophobic domain of C8α-γ to insert into the lipid bilayer. Finally, C8α-γ induces the polymerization of 10–16 molecules of C9 into a pore-forming structure called the membrane-attack complex. The membrane-attack complex has a hydrophobic external face, allowing it to associate with the lipid bilayer, but a hydrophilic internal channel. The diameter of this channel is about 10 nm, allowing the free passage of solutes and water across the lipid bilayer. The pore damage to the lipid bilayer leads to the loss of cellular homeostasis, the disruption of the proton gradient across the membrane, the penetration of enzymes such as lysozyme into the cell, and the eventual destruction of the pathogen.


Immunology_Janeway

Immunology_Janeway. Although the effect of the membrane-attack complex is very dramatic, particularly in experimental demonstrations in which antibodies against red blood cell membranes are used to trigger the complement cascade, the significance The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. Fig. 2.35 Assembly of the membrane-attack complex polymerization of C9, again with the exposure of a hydrophobic generates a pore in the lipid bilayer membrane. The sequence site. Up to 16 molecules of C9 are then added to the assembly of steps and their approximate appearance are shown here in to generate a channel 10 nm in diameter in the membrane. This schematic form. C5b triggers the assembly of a complex of channel disrupts the bacterial cell membrane, killing the bacterium. one molecule each of C6, C7, and C8, in that order. C7 and C8 The electron micrographs show erythrocyte membranes with undergo conformational changes, exposing hydrophobic domains membrane-attack complexes in two orientations, end on and side that insert into the membrane. This complex causes moderate on. Photographs courtesy of S. Bhakdi and J. Tranum-Jensen. membrane damage in its own right, and also serves to induce the of these components in host defense seems to be quite limited. So far, deficiencies in


Immunology_Janeway

Immunology_Janeway. courtesy of S. Bhakdi and J. Tranum-Jensen. membrane damage in its own right, and also serves to induce the of these components in host defense seems to be quite limited. So far, deficiencies in complement components C5–C9 have been associated with susceptibility only to Neisseria species, the bacteria that cause the sexually transmitted disease gonorrhea and a common form of bacterial meningitis. Thus, the opsonizing and inflammatory actions of the earlier components of the complement cascade are clearly more important for host defense against infection. Formation of the membrane-attack complex seems to be important only for the killing of a few pathogens, although, as we will see in Chapter 15, this complex might well have a major role in immunopathology.


Immunology_Janeway

Immunology_Janeway. of complement activation and protect the host from their destructive effects. Complement activation usually is initiated on a pathogen surface, and the activated complement fragments that are produced usually bind nearby on the pathogen surface or are rapidly inactivated by hydrolysis. Even so, all complement components are activated spontaneously at a low rate in plasma, and these activated complement components will sometimes bind proteins on host cells. Section 2-10 introduced the soluble host proteins factor I and factor H and the membrane-bound proteins MCP and DAF that regulate the alternative pathway of complement activation. In addition to these, several other soluble and membrane-bound complement-control proteins can regulate the complement cascade at various steps to protect normal host cells while allowing complement activation to proceed on pathogen surfaces (Fig. 2.36).


Immunology_Janeway

Immunology_Janeway. The activation of C1 is controlled by the C1 inhibitor (C1INH), which is a plasma serine protease inhibitor, or serpin. C1INH binds to the active enzymes C1r:C1s and causes them to dissociate from C1q, which remains bound to the pathogen (Fig. 2.37). In this way, C1INH limits the time during which active C1s is able to cleave C4 and C2. By the same means, C1INH limits the spontaneous activation of C1 in plasma. Its importance can be seen in the C1INH deficiency disease hereditary angioedema (HAE), in which chronic spontaneous complement activation leads to the production of excess cleaved fragments of C4 and C2. The large activated fragments from this cleavage, which normally combine to form the C3 convertase, do not damage host cells in such patients because C4b is rapidly inactivated by hydrolysis in plasma, The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. The complement system and innate immunity. Fig. 2.37 Complement activation is regulated by a series of proteins that serve to protect host cells from accidental damage. These act on different stages of the complement cascade, dissociating complexes or catalyzing the enzymatic degradation of covalently bound complement proteins. Stages in the complement cascade are shown schematically down the left side of the figure, with the regulatory reactions on the right. The alternative-pathway C3 convertase is similarly regulated by DAF, CR1, MCP, and factor H.


Immunology_Janeway

Immunology_Janeway. and the convertase does not form. However, the small fragment of C2, C2b, is further cleaved into a peptide, the C2 kinin, that causes extensive swell-ing—the most dangerous being local swelling in the larynx that can lead to suffocation. Bradykinin, which has similar actions to those of C2 kinin, is also produced in an uncontrolled fashion in this disease, as a result of the lack of inhibition of kallikrein, another plasma protease and component of the kinin system (discussed in Section 3-3). Kallikrein is activated by tissue damage and is also regulated by C1INH. Hereditary angioedema is fully corrected by replacing C1INH. A similar, extremely rare human disease stems from a partial deficiency of carboxypeptidase N (CPN), a metalloproteinase that inactivates the anaphylatoxins C3a and C5a as well as bradykinin and kallikrein. Humans with partial CPN deficiency exhibit recurrent angioedema due to delayed inactivation of serum C3a and bradykinin.


Immunology_Janeway

Immunology_Janeway. Since the highly reactive thioester bond of activated C3 and C4 cannot distinguish between acceptor groups on a host cell from those on the surface of a pathogen, mechanisms have evolved that prevent the small amounts of C3 or C4 molecules deposited on host cells from fully triggering complement activation. We introduced these mechanisms in the context of control of the alternative pathway (see Fig. 2.27), but they are also important regulators of the classical pathway convertase (see Fig. 2.37, second and third rows). Section 2-10 described the proteins that inactivate any C3b or C4b that has bound to host cells. These are the plasma factor I and its cofactors MCP and CR1, which are membrane proteins. Circulating factor I is an active serine protease, but it can cleave C3b and C4b only when they are bound to MCP and CR1. In these circumstances, factor I cleaves C3b, first into iC3b and then further to C3dg, thus permanently inactivating it. C4b is similarly inactivated by cleavage


Immunology_Janeway

Immunology_Janeway. when they are bound to MCP and CR1. In these circumstances, factor I cleaves C3b, first into iC3b and then further to C3dg, thus permanently inactivating it. C4b is similarly inactivated by cleavage into C4c and C4d. Microbial cell walls lack MCP and CR1 and thus cannot promote the breakdown of C3b and C4b, which instead act as binding sites for factor B and C2, promoting complement activation. The importance of factor I can be seen in people with genetically determined factor I deficiency. Because of uncontrolled complement activation, complement proteins rapidly become depleted and such people suffer repeated bacterial infections, especially with ubiquitous pyogenic bacteria.


Immunology_Janeway

Immunology_Janeway. There are also plasma proteins with cofactor activity for factor I, most notably C4b-binding protein (C4BP) (see Fig. 2.36). It binds C4b and acts mainly as a regulator of the classical pathway in the fluid phase. Another is factor H, which binds C3b in the fluid phase as well as at cell membranes and helps to distinguish the C3b that is bound to host cells from that bound to microbial surfaces. The higher affinity of factor H for sialic acid residues on host membrane glycoproteins allows it to displace factor B in binding to C3b on host cells. Also, C3b at cell membranes is bound by cofactor proteins DAF and MCP. Factor H, DAF, and MCP effectively compete with factor B for binding to C3b bound to host cells, so that the bound C3b is catabolized by factor I into iC3b and C3dg and complement activation is inhibited. In contrast, factor B is favored for binding C3b on microbial membranes, which do not express DAF or MCP and which lack the sialic acid modifications that attract factor H.


Immunology_Janeway

Immunology_Janeway. activation is inhibited. In contrast, factor B is favored for binding C3b on microbial membranes, which do not express DAF or MCP and which lack the sialic acid modifications that attract factor H. The greater amount of factor B on a microbial surface stimulates formation of more C3bBb C3 convertase, and thus complement activation is amplified.


Immunology_Janeway

Immunology_Janeway. The critical balance between the inhibition and the activation of complement on cell surfaces is illustrated in individuals heterozygous for mutations in any of the regulatory proteins MCP, factor I, or factor H. In such individuals, the concentration of functional regulatory proteins is reduced, and the tipping of the balance toward complement activation leads to a predisposition to atypical hemolytic uremic syndrome, a condition characterized by damage to platelets and red blood cells and by kidney inflammation. Another serious health problem related to complement malfunction is a significantly increased The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. The complement system and innate immunity. risk of age-related macular degeneration, the leading cause of blindness in the elderly in developed countries, which has been predominantly linked to single-nucleotide polymorphisms in the factor H gene. Polymorphisms in other complement genes have also been found to be either detrimental or protective for this disease. Thus, even small alterations in the efficiency of either the activation or the regulation of this powerful effector system can contribute to the progression of degenerative or inflammatory disorders.


Immunology_Janeway

Immunology_Janeway. The competition between DAF or MCP and factor B for binding to surface-bound C3b is an example of the second mechanism for inhibiting complement activation on host cells. By binding C3b and C4b on the cell surface, these proteins competitively inhibit the binding of C2 to cell-bound C4b and of factor B to cell-bound C3b, thereby inhibiting convertase formation. DAF and MCP also mediate protection against complement through a third mechanism, which is to augment the dissociation of C4b2a and C3bBb convertases that have already formed. Like DAF, CR1 is among the host-cell membrane molecules that regulate complement through both these mechanisms—that is, by promoting the dissociation of convertase and exhibiting cofactor activity. All the proteins that bind the homologous C4b and C3b molecules share one or more copies of a structural element called the short consensus repeat (SCR), the complement control protein (CCP) repeat, or (especially in Japan) the sushi domain.


Immunology_Janeway

Immunology_Janeway. In addition to the mechanisms for preventing C3 convertase formation and C4 and C3 deposition on cell membranes, there are also inhibitory mechanisms that prevent the inappropriate insertion of the membrane-attack complex (MAC) into membranes. We saw in Section 2-15 that the membrane-attack complex polymerizes onto C5b molecules created by the action of C5 convertase. The MAC complex mainly inserts into cell membranes adjacent to the site of the C5 convertase, that is, close to the site of complement activation on a pathogen. However, some newly formed membrane-attack complexes may diffuse from the site of complement activation and insert into adjacent host-cell membranes. Several plasma proteins, including, notably, vitronectin (also known as S-protein), bind to the C5b67, C5b678, and C5b6789 complexes and thereby inhibit their random insertion into cell membranes. Host-cell membranes also contain an intrinsic protein, CD59, or protectin, that inhibits the binding of C9 to the C5b678


Immunology_Janeway

Immunology_Janeway. complexes and thereby inhibit their random insertion into cell membranes. Host-cell membranes also contain an intrinsic protein, CD59, or protectin, that inhibits the binding of C9 to the C5b678 complex (see Fig. 2.37, bottom row). CD59 and DAF are both linked to the cell surface by a glycosylphosphatidylinositol (GPI) tail, like many other peripheral membrane proteins. One of the enzymes involved in the synthesis of GPI tails is encoded by a gene, PIGA, on the X chromosome. In people with a somatic mutation in this gene in a clone of hematopoietic cells, both CD59 and DAF fail to function. This causes the disease paroxysmal nocturnal hemoglobinuria, which is characterized by episodes of intravascular red blood cell lysis by complement. Red blood cells that lack only CD59 are also susceptible to destruction as a result of spontaneous activation of the complement cascade.


Immunology_Janeway

Immunology_Janeway. 2-17 Pathogens produce several types of proteins that can inhibit complement activation. Bacterial pathogens have evolved various strategies to avoid activation of complement, and thereby to avoid elimination by this first line of innate defense (Fig. 2.38). One strategy that many pathogens employ is to mimic host surfaces by attracting host complement regulators to their own surfaces. A mechanism to achieve this is for the pathogen to express surface proteins that bind to soluble complement-regulatory proteins such as C4BP and factor H. For example, the Gram-negative pathogen Neisseria meningitidis produces factor H binding protein (fHbp), which recruits factor H (see Section 2-10), and the outer Fig. 2.38 Complement evasion proteins produced by various pathogens.


Immunology_Janeway

Immunology_Janeway. Fig. 2.38 Complement evasion proteins produced by various pathogens. membrane protein PorA, which binds to C4BP. By recruiting factor H and C4BP to the pathogen membrane, the pathogen is able to inactivate C3b that is deposited on its surface and thereby avoid the consequences of complement activation. Complement is important in defense against Neisseria species, and several complement deficiencies are associated with increased susceptibility to this pathogen.


Immunology_Janeway

Immunology_Janeway. Another strategy employed by pathogens is to secrete proteins that directly inhibit components of complement. The Gram-positive pathogen Staphylococcus aureus provides several examples of this type of strategy. Staphylococcal protein A (Spa) binds to the Fc regions of immunoglobulins and interferes with the recruitment and activation of C1. This binding specificity was used as an early biochemical technique in the purification of antibodies. The staphylococcal protein staphylokinase (SAK) acts by cleaving immunoglobulins bound to the pathogen membrane, preventing complement activation and avoiding phagocytosis. The staphylococcal complement inhibitor (SCIN) protein binds to the classical C3 convertase, C4b2a, and the alternative pathway C3 convertase, C3bBb, and inhibits their activity. Other stages of complement activation, including formation of the C5 convertase, are targets of inhibition by proteins produced by these and other pathogens. We will return to this topic of complement


Immunology_Janeway

Immunology_Janeway. stages of complement activation, including formation of the C5 convertase, are targets of inhibition by proteins produced by these and other pathogens. We will return to this topic of complement regulation in Chapter 13 when we discuss how the immune system sometimes fails or is evaded by pathogens.


Immunology_Janeway

Immunology_Janeway. Summary. The complement system is one of the major mechanisms by which pathogen recognition is converted into an effective host defense against initial infection. Complement is a system of plasma proteins that can be activated directly by pathogens or indirectly by pathogen-bound antibody, leading to a cascade of The complement system and innate immunity.


Immunology_Janeway

Immunology_Janeway. reactions that occurs on the surface of pathogens and generates active components with various effector functions. There are three pathways of complement activation: the lectin pathway, triggered by the pattern recognition receptors MBL and the ficolins; the classical pathway, triggered directly by antibody binding to the pathogen surface; and the alternative pathway, which utilizes spontaneous C3 deposition onto microbial surfaces, is augmented by proper-din, and provides an amplification loop for the other two pathways. The early events in all pathways consist of a sequence of cleavage reactions in which the larger cleavage product binds covalently to the pathogen surface and contributes to the activation of the next component. The pathways converge with the formation of a C3 convertase enzyme, which cleaves C3 to produce the active complement component C3b. The binding of large numbers of C3b molecules to the pathogen is the central event in complement activation. Bound complement


Immunology_Janeway

Immunology_Janeway. enzyme, which cleaves C3 to produce the active complement component C3b. The binding of large numbers of C3b molecules to the pathogen is the central event in complement activation. Bound complement components, especially bound C3b and its inactive fragments, are recognized by specific complement receptors on phagocytic cells, which engulf pathogens opsonized by C3b and its inactive fragments. The small cleavage fragments of C3 and C5 act on specific trimeric G-protein-coupled receptors to recruit phagocytes, such as neutrophils, to sites of infection. Together, these activities promote the uptake and destruction of pathogens by phagocytes. The molecules of C3b that bind the C3 convertase itself initiate the late events of complement, binding C5 to make it susceptible to cleavage by C2a or Bb. The larger C5b fragment triggers the assembly of a membrane-attack complex, which can result in the lysis of certain pathogens. A system of soluble and membrane-bound complement-regulatory


Immunology_Janeway

Immunology_Janeway. C2a or Bb. The larger C5b fragment triggers the assembly of a membrane-attack complex, which can result in the lysis of certain pathogens. A system of soluble and membrane-bound complement-regulatory proteins act to limit complement activation on host tissues, in order to prevent tissue damage from the inadvertent binding of activated complement components to host cells or from the spontaneous activation of complement components in plasma. Many pathogens produce a variety of soluble and membrane-associated proteins that can counteract complement activation and contribute to infection of the host by the microbe.


Immunology_Janeway

Summary to Chapter 2.

Immunology_Janeway. Summary to Chapter 2.