Chapter 2:  Innate Immunity

2-1. Infectious agents must overcome innate host defenses to establish a focus of infection

Our bodies are constantly exposed to microorganisms present in the environment, including infectious agents that have been shed from infected individuals. Contact with these microorganisms may occur through external or internal epithelial surfaces: the respiratory tract mucosa provides a route of entry for airborne microorganisms, the gastrointestinal mucosa for microorganisms in food and water; insect bites and wounds allow micro-organisms to penetrate the skin; and direct contact between individuals offers opportunities for infection of the skin and reproductive mucosa (Fig. 2.2).

In spite of this exposure, infectious disease is fortunately quite rare. The epithelial surfaces of the body serve as an effective barrier against most microorganisms, and are rapidly repaired if wounded. Furthermore, most of the microorganisms that do succeed in crossing the epithelial surfaces are efficiently removed by innate immune mechanisms that function in the underlying tissues. Thus in most cases these defenses, which we will examine in more detail in subsequent sections, prevent a site of infection from being established. It is difficult to know how many infections are repelled in this way, because there are no symptoms of disease. It is clear, however, that the microorganisms that a normal human being inhales or ingests, or that enter through minor wounds, are mostly held at bay or eliminated, since they seldom cause disease.

Infectious disease occurs when a microorganism succeeds in evading or overwhelming innate host defenses to establish a local site of infection and replication that allows its further transmission. In some cases, such as athlete’s foot, the initial infection remains local and does not cause significant pathology. In other cases the infectious agent causes significant pathology as it spreads through the lymphatics or the bloodstream, or as a result of secreting toxins.

Pathogen spread is often countered by an inflammatory response that recruits more effector molecules and cells of the innate immune system from local blood vessels (Fig. 2.3), while inducing clotting farther downstream so that pathogens cannot spread through the blood. The induced responses of innate immunity act over several days while an adaptive immune response gets underway in response to pathogen antigens delivered to local lymphoid tissue. Such a response can target specific features of the pathogen and will usually clear the infection and protect the host against reinfection with the same pathogen.

2-2. The epithelial surfaces of the body are the first defenses against infection

Our body surfaces are defended by epithelia, which provide a physical barrier between the internal milieu and the external world that contains pathogens. Epithelial cells are held together by tight junctions, which effectively form a seal against the external environment. Epithelia comprise the skin and the linings of the body’s tubular structures—the gastrointestinal, respiratory, and urinogenital tracts. Infections occur only when the pathogen can colonize or cross through these barriers, and since the dry, protective layers of the skin present a more formidable barrier, pathogen entry most often occurs through the internal epithelial surfaces. The importance of epithelia in protection against infection is obvious when the barrier is breached, as in wounds and burns, when infection is a major cause of mortality and morbidity. In the absence of wounding or disruption, pathogens normally cross epithelial barriers by binding to molecules on internal epithelial surfaces, or establish an infection by adhering to and colonizing these surfaces. This specific attachment allows the pathogen to infect the epithelial cell, or to damage it so that the epithelium can be crossed, or, in the case of colonizing pathogens, to avoid being dislodged by the flow of air or fluid across the epithelial surface. The internal epithelia are known as mucosal epithelia because they secrete a viscous fluid called mucus, which contains many glycoproteins called mucins. Microorganisms coated in mucus may be prevented from adhering to the epithelium, and in mucosal epithelia such as that of the respiratory tract, microorganisms can be expelled in the flow of mucus driven by the beating of epithelial cilia. The efficacy of mucus flow in clearing infection is illustrated by people with defective mucus secretion or inhibition of ciliary movement; they frequently develop lung infections caused by bacteria that colonize the epithelial surface. In the gut, peristalsis is an important mechanism for keeping both food and infectious agents moving through. Failure of peristalsis is typically accompanied by overgrowth of bacteria within the intestinal lumen.

Our surface epithelia are more than mere physical barriers to infection; they also produce chemical substances that are microbicidal or inhibit microbial growth (Fig. 2.4). For example, the antibacterial enzyme lysozyme is secreted in tears and saliva. The acid pH of the stomach and the digestive enzymes of the upper gastrointestinal tract create a substantial chemical barrier to infection. Further down the intestinal tract, antibacterial and antifungal peptides called cryptidins or α-defensins are made by Paneth cells, which are resident in the base of the crypts in the small intestine beneath the epithelial stem cells. Related antimicrobial peptides, the β-defensins, are made by other epithelia, primarily in the skin and respiratory tract. Such antimicrobial peptides play a role in the immune defense of many organisms, including insects. They are cationic peptides that are thought to kill bacteria by damaging the bacterial cell membrane. Another type of antimicrobial protein is secreted into the fluid that bathes the epithelial surfaces of the lung. This fluid contains two proteins—surfactant proteins A and D—that bind to and coat the surfaces of pathogens so that they are more easily phagocytosed by macrophages that have left the subepithelial tissues to enter the alveoli of the lung. Coating of a particle with proteins that facilitate its phagocytosis is known as opsonization and we will meet several examples of this defense strategy in this chapter.

In addition to these defenses, most epithelial surfaces are associated with a normal flora of nonpathogenic bacteria that compete with pathogenic microorganisms for nutrients and for attachment sites on cells. The normal flora can also produce antimicrobial substances, such as the colicins (anti-bacterial proteins made by Escherichia coli) that prevent colonization by other bacteria. When the nonpathogenic bacteria are killed by antibiotic treatment, pathogenic microorganisms frequently replace them and cause disease.

2-3. After entering tissues, many pathogens are recognized, ingested, and killed by phagocytes

If a microorganism crosses an epithelial barrier and begins to replicate in the tissues of the host, it is, in most cases, immediately recognized by the mononuclear phagocytes, or macrophages, that reside in tissues. Macrophages mature continuously from circulating monocytes that leave the circulation to migrate into tissues throughout the body (see Fig. 1.3). They are found in especially large numbers in connective tissue, in association with the gastrointestinal tract, in the lung (where they are found in both the interstitium and the alveoli), along certain blood vessels in the liver (where they are known as Kupffer cells), and throughout the spleen, where they remove senescent blood cells. The second major family of phagocytes—the neutrophils, or polymorphonuclear neutrophilic leukocytes (PMNs or polys)—are short-lived cells that are abundant in the blood but are not present in normal, healthy tissues. Both these phagocytic cells have a key role in innate immunity because they can recognize, ingest, and destroy many pathogens without the aid of an adaptive immune response. Macrophages are the first to encounter pathogens in the tissues but they are soon re-inforced by the recruitment of large numbers of neutrophils to sites of infection.

Macrophages and neutrophils recognize pathogens by means of cell-surface receptors that can discriminate between the surface molecules displayed by pathogens and those of the host. These receptors, which we will examine in more detail later, include the macrophage mannose receptor, which is found on macrophages but not on monocytes or neutrophils, scavenger receptors, which bind many charged ligands, and CD14, a receptor for bacterial lipopolysaccharide (LPS) found predominantly on monocytes and macro-phages (Fig. 2.5). Pathogens can also interact with macrophages and neutrophils through receptors for complement borne on these cells. As we will see in the second part of the chapter, the complement system is activated rapidly in response to many types of infection, producing complement proteins that opsonize the surface of pathogens as they enter the tissues.

Ligation of many of the cell-surface receptors that recognize pathogens leads to phagocytosis of the pathogen, followed by its death inside the phagocyte. Phagocytosis is an active process, in which the bound pathogen is first surrounded by the phagocyte membrane and then internalized in a membrane-bounded vesicle known as a phagosome, which becomes acidified. In addition to being phagocytic, macrophages and neutrophils have granules, called lysosomes, that contain enzymes, proteins, and peptides that can mediate an intracellular antimicrobial response. The phagosome fuses with one or more lysosomes to generate a phagolysosome in which the lysosomal contents are released to destroy the pathogen (see Fig. 2.5).

Upon phagocytosis, macrophages and neutrophils also produce a variety of other toxic products that help kill the engulfed microorganism (Fig. 2.6). The most important of these are hydrogen peroxide (H₂O₂), the superoxide anion (O₂^–), and nitric oxide (NO), which are directly toxic to bacteria. They are generated by lysosomal NADPH oxidases and other enzymes in a process known as the respiratory burst, as it is accompanied by a transient increase in oxygen consumption. Neutrophils are short-lived cells, dying soon after they have accomplished a round of phagocytosis. Dead and dying neutrophils are a major component of the pus that forms in some infections; bacteria that give rise to such infections are thus known as pyogenic bacteria. Macrophages, on the other hand, are long-lived and continue to generate new lysosomes. Patients with chronic granulomatous disease have a genetic deficiency of NADPH oxidase, which means that their phagocytes do not produce toxic oxygen derivatives and are less able to kill ingested microorganisms and clear the infection. People with this defect are unusually susceptible to bacterial and fungal infections, especially in infancy.

Macrophages can make this response immediately on encountering an infecting microorganism and this can be sufficient to prevent an infection from becoming established. The great cellular immunologist Elie Metchnikoff believed that the innate response of macrophages encompassed all host defense and, indeed, it is now clear that invertebrates, such as the sea star that he was studying, rely entirely on innate immunity for their defense against infection. Although this is not the case in humans and other vertebrates, the innate response of macrophages still provides an important front line of host defense that must be overcome if a microorganism is to establish an infection that can be passed on to a new host.

A key feature that distinguishes pathogenic from nonpathogenic micro-organisms is their ability to overcome innate immune defenses. Pathogens have developed a variety of strategies to avoid being immediately destroyed by macrophages. Many extracellular pathogenic bacteria coat themselves with a thick polysaccharide capsule that is not recognized by any phagocyte receptor. Other pathogens, for example mycobacteria, have evolved ways to grow inside macrophage phagosomes by inhibiting fusion with a lysosome. Without such devices, a microorganism must enter the body in sufficient numbers to simply overwhelm the immediate innate host defenses and establish a focus of infection.

The second important effect of the interaction between pathogens and tissue macrophages is activation of macrophages to release cytokines and other mediators that set up a state of inflammation in the tissue and bring neutrophils and plasma proteins to the site of infection. It is thought that the pathogen induces cytokine secretion by signals delivered through some of the receptors to which it binds, and we will see later how this occurs in response to LPS. Receptors that signal the presence of pathogens and induce cytokines also have another important role. This is to induce the expression of so-called co-stimulatory molecules on both macrophages and dendritic cells, another type of phagocytic cell present in tissues, thus enabling these cells to initiate an adaptive immune response (see Section 1-6).

The cytokines released by macrophages make an important contribution both to local inflammation and to other induced but nonadaptive responses that occur in the first few days of a new infection. We will be describing the role of individual cytokines in these induced responses in the last part of this chapter. However, since an inflammatory response is usually initiated within minutes of infection or wounding, we will outline here how it occurs and how it contributes to host defense.

2-4. Pathogen recognition and tissue damage initiate an inflammatory response

Inflammation plays three essential roles in combating infection. The first is to deliver additional effector molecules and cells to sites of infection to augment the killing of invading microorganisms by the front-line macrophages. The second is to provide a physical barrier preventing the spread of infection, and the third is to promote the repair of injured tissue, a nonimmunological role that we will not discuss further. Inflammation at the site of infection is initiated by the response of macrophages to pathogens.

Inflammatory responses are operationally characterized by pain, redness, heat, and swelling at the site of an infection, reflecting three types of change in the local blood vessels. The first is an increase in vascular diameter, leading to increased local blood flow—hence the heat and redness—and a reduction in the velocity of blood flow, especially along the surfaces of small blood vessels. The second change is that the endothelial cells lining the blood vessel are activated to express adhesion molecules that promote the binding of circulating leukocytes. The combination of slowed blood flow and induced adhesion molecules allows leukocytes to attach to the endothelium and migrate into the tissues, a process known as extravasation, which we will describe in detail later. All these changes are initiated by the cytokines produced by activated macrophages. Once inflammation has begun, the first cells attracted to the site of infection are generally neutrophils. They are followed by monocytes, which differentiate into more tissue macrophages. In the later stages of inflammation, other leukocytes such as eosinophils and lymphocytes also enter the infected site. The third major change in the local blood vessels is an increase in vascular permeability. Instead of being tightly joined together, the endothelial cells lining the blood vessel walls become separated, leading to exit of fluid and proteins from the blood and their local accumulation in the tissue. This accounts for the swelling, or edema, and pain—as well as the accumulation of plasma proteins that aid in host defense.

These changes are induced by a variety of inflammatory mediators released as a consequence of the recognition of pathogens. These include the lipid mediators of inflammation—prostaglandins, leukotrienes, and platelet-activating factor (PAF)—which are rapidly produced by macrophages through enzymatic pathways that degrade membrane phospholipids. Their actions are followed by those of the cytokines and chemokines (chemoattractant cytokines) that are synthesized and secreted by macrophages in response to pathogens. The cytokine tumor necrosis factor-α (TNF-α), for example, is a potent activator of endothelial cells.

As we will see in the next part of the chapter, another way in which pathogen recognition rapidly triggers an inflammatory response is through activation of the complement cascade. One of the cleavage products of the complement reaction is a peptide called C5a. C5a is a potent mediator of inflammation, with several different activities. In addition to increasing vascular permeability and inducing the expression of some adhesion molecules, it acts as a powerful chemoattractant for neutrophils and monocytes, and activates phagocytes and local mast cells, which are in turn stimulated to release granules containing the inflammatory molecule histamine and TNF-α.

If wounding has occurred, the injury to blood vessels immediately triggers two other protective enzyme cascades. The kinin system is an enzymatic cascade of plasma proteins that is triggered by tissue damage to produce several inflammatory mediators, including the vasoactive peptide bradykinin. This causes an increase in vascular permeability that promotes the influx of plasma proteins to the site of tissue injury. It also causes pain, which, although unpleasant to the victim, draws attention to the problem and leads to immobilization of the affected part of the body, which helps to limit the spread of any infectious agents. The coagulation system is another enzymatic cascade of plasma enzymes that is triggered following damage to blood vessels. This leads to the formation of a clot, which prevents any microorganisms from entering the bloodstream. Both these cascades have an important role in the inflammatory response to pathogens even if wounding or gross tissue injury has not occurred, as they are also triggered by endothelial cell activation. Thus, within minutes of the penetration of tissues by a pathogen, the inflammatory response causes an influx of proteins and cells that will control the infection. It also forms a physical barrier to limit the spread of infection and makes the host fully aware of what is going on.

Summary

The mammalian body is susceptible to infection by many pathogens, which must first make contact with the host and then establish a focus of infection in order to cause disease. These pathogens differ greatly in their lifestyles, the structures of their surfaces, and means of pathogenesis, which therefore requires an equally diverse set of defensive responses from the host immune system. The first phase of host defense consists of those mechanisms that are present and ready to resist an invader at any time. The epithelial surfaces of the body keep pathogens out, and protect against colonization and against viruses and bacteria that enter through specialized cell-surface interactions, by preventing pathogen adherence and by secreting antimicrobial enzymes and peptides. Bacteria, viruses, and parasites that overcome this barrier are faced immediately by tissue macrophages equipped with surface receptors that can bind and phagocytose many different types of pathogen. This, in turn, leads to an inflammatory response, which causes the accumulation of plasma proteins, including the complement components that provide circulating or humoral innate immunity, as will be described in the next part of the chapter, and phagocytic neutrophils at the site of infection. Innate immunity provides a front line of host defense through effector mechanisms that engage the pathogen directly, act immediately on contact with it, and are unaltered in their ability to resist a subsequent challenge with either the same or a different pathogen. These mechanisms often succeed in preventing an infection from becoming established. If not, they are reinforced through the recruitment and increased production of further effector molecules and cells in a series of induced responses that we will consider later in this chapter. These induced innate responses often fail to clear the infection. In that case, macrophages and other cells activated in the early innate response help to initiate the development of an adaptive immune response.

2-5. Complement is a system of plasma proteins that interacts with pathogens to mark them for destruction by phagocytes

In the early phases of an infection, the complement cascade can be activated on the surface of a pathogen through any one, or more, of the three pathways shown in Fig. 2.8. The classical pathway can be initiated by the binding of C1q, the first protein in the complement cascade, directly to the pathogen surface. It can also be activated during an adaptive immune response by the binding of C1q to antibody:antigen complexes, and is thus a key link between the effector mechanisms of innate and adaptive immunity. The mannan-binding lectin pathway (MB-lectin pathway) is initiated by binding of the mannan-binding lectin, a serum protein, to mannose-containing carbohydrates on bacteria or viruses. Finally, the alternative pathway can be initiated when a spontaneously activated complement component binds to the surface of a pathogen. Each pathway follows a sequence of reactions to generate a protease called a C3 convertase. These reactions are known as the ‘early’ events of complement activation, and consist of triggered-enzyme cascades in which inactive complement zymogens are successively cleaved to yield two fragments, the larger of which is an active serine protease. The active protease is retained at the pathogen surface, and this ensures that the next complement zymogen in the pathway is also cleaved and activated at the pathogen surface. By contrast, the small peptide fragment is released from the site of the reaction and can act as a soluble mediator.

The C3 convertases formed by these early events of complement activation are bound covalently to the pathogen surface. Here they cleave C3 to generate large amounts of C3b, the main effector molecule of the complement system, and C3a, a peptide mediator of inflammation. The C3b molecules act as opsonins; they bind covalently to the pathogen and thereby target it for destruction by phagocytes equipped with receptors for C3b. C3b also binds the C3 convertase to form a C5 convertase that produces the most important small peptide mediator of inflammation, C5a, as well as a large active fragment, C5b, that initiates the ‘late’ events of complement activation. These comprise a sequence of polymerization reactions in which the terminal complement components interact to form a membrane-attack complex, which creates a pore in the cell membranes of some pathogens that can lead to their death.

The nomenclature of complement proteins is often a significant obstacle to understanding this system, and before discussing the complement cascade in more detail, we will explain the conventions, and the nomenclature used in this book. All components of the classical complement pathway and the membrane-attack complex are designated by the letter C followed by a number. The native components have a simple number designation, for example, C1 and C2, but unfortunately, the components were numbered in the order of their discovery rather than the sequence of reactions, which is C1, C4, C2, C3, C5, C6, C7, C8, and C9. The products of the cleavage reactions are designated by added lower-case letters, the larger fragment being designated b and the smaller a; thus, for example, C4 is cleaved to C4b, the large fragment of C4 that binds covalently to the surface of the pathogen, and C4a, a small fragment with weak pro-inflammatory properties. The components of the alternative pathway, instead of being numbered, are designated by different capital letters, for example factor B and factor D. As with the classical pathway, their cleavage products are designated by the addition of lower-case a and b: thus, the large fragment of B is called Bb and the small fragment Ba. Finally, in the mannose-binding lectin pathway, the first enzymes to be activated are known as the mannan-binding lectin-associated serine proteases MASP-1 and MASP-2, after which the pathway is essentially the same as the classical pathway. Activated complement components are often designated by a horizontal line, for example, ; however, we will not use this convention. It is also useful to be aware that the large active fragment of C2 was originally designated C2a, and is still called that in some texts and research papers. Here, for consistency, we will call all large fragments of complement b, so the large active fragment of C2 will be designated C2b.

The formation of C3 convertase activity is pivotal in complement activation, leading to the production of the principal effector molecules, and initiating the late events. In the classical and MB-lectin pathways, the C3 convertase is formed from membrane-bound C4b complexed with C2b. In the alternative pathway, a homologous C3 convertase is formed from membrane-bound C3b complexed with Bb. The alternative pathway can act as an amplification loop for all three pathways, as it is initiated by the binding of C3b.

It is clear that a pathway leading to such potent inflammatory and destructive effects, and which, moreover, has a series of built-in amplification steps, is potentially dangerous and must be subject to tight regulation. One important safeguard is that 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 on complement components to prevent the inadvertent activation of complement on host cell surfaces, hence protecting them from accidental damage. We will return to these regulatory mechanisms later.

We have now introduced all the relevant components of complement and are ready for a more detailed account of their functions. To help distinguish the different components according to their functions, we will use a color code in the figures in this part of the chapter. This is introduced in Fig. 2.9, where all the components of complement are grouped by function.

2-6. The classical pathway is initiated by activation of the C1 complex

The classical pathway plays a role in both innate and adaptive immunity. As we will see in Chapter 9, the first component of this pathway, C1q, links the adaptive humoral immune response to the complement system by binding to antibodies complexed with antigens. C1q can, however, also bind directly to the surface of certain pathogens and thus trigger complement activation in the absence of antibody. C1q is part of the C1 complex, which comprises a single C1q molecule bound to two molecules each of the zymogens C1r and C1s. C1q is a calcium-dependent sugar-binding protein, a lectin, belonging to the collectin family of proteins, which contains both collagen-like and lectin domains hence the name collectin. It has six globular heads, linked together by a collagen-like tail, which surround the (C1r:C1s)₂ complex (Fig. 2.10). Binding of more than one of the C1q heads to a pathogen surface causes a conformational change in the (C1r:C1s)₂ complex, which leads to activation of an autocatalytic enzymatic activity in C1r; the active form of C1r then cleaves its associated C1s to generate an active serine protease.

Once activated, the C1s enzyme acts on the next two components of the classical pathway, cleaving C4 and then C2 to generate two large fragments, C4b and C2b, which together form the C3 convertase of the classical pathway. In the first step, C1s cleaves C4 to produce C4b, which binds covalently to the surface of the pathogen. The covalently attached C4b then binds one molecule of C2, making it susceptible, in turn, to cleavage by C1s. C1s cleaves C2 to produce the large fragment C2b, which is itself a serine protease. The complex of C4b with the active serine protease C2b remains on the surface of the pathogen as the C3 convertase of the classical pathway. Its most important activity is to cleave large numbers of C3 molecules to produce C3b molecules that coat the pathogen surface. At the same time, the other cleavage product, C3a, initiates a local inflammatory response. These reactions, which comprise the classical pathway of complement activation, are shown in schematic form in Fig. 2.11; the proteins involved, and their active forms, are listed in Fig. 2.12.

2-7. The mannan-binding lectin pathway is homologous to the classical pathway

The MB-lectin pathway uses a protein very similar to C1q to trigger the complement cascade. This protein, called the mannan-binding lectin (MBL), is a collectin, like C1q. Mannan-binding lectin binds specifically to mannose residues, and to certain other sugars, which are accessible and arranged in a pattern that allows binding on many pathogens. On vertebrate cells, however, these are covered by other sugar groups, especially sialic acid. Thus, mannan-binding lectin is able to initiate complement activation by binding to pathogen surfaces. It is present at low concentrations in normal plasma of most individuals, and, as we will see in the last part of this chapter, its production by the liver is increased during the acute-phase reaction of the innate immune response.

Mannan-binding lectin, like C1q, is a six-headed molecule that forms a complex with two protease zymogens, which in the case of the mannanbinding lectin complex (MBL complex) are MASP-1 and MASP-2 (Fig. 2.13). MASP-1 and MASP-2 are closely homologous to C1r and C1s, and all four enzymes are likely to have evolved from gene duplication of a common precursor. When the MBL complex binds to a pathogen surface, MASP-1 and MASP-2 are activated to cleave C4 and C2. Thus the MB-lectin pathway initiates complement activation in the same way as the classical pathway, forming a C3 convertase from C2b bound to C4b. People deficient in mannan-binding lectin experience a substantial increase in infections during early childhood, indicating the importance of the MB-lectin pathway for host defense. The age window of susceptibility to infections associated with mannan-binding lectin deficiency illustrates the particular importance of innate host defense mechanisms in childhood, before the child’s adaptive immune responses are fully matured and after maternal antibodies transferred across the placenta and in colostrum have been lost.

2-8. Complement activation is largely confined to the surface on which it is initiated

We have seen that the classical and MB-lectin 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 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. Cleavage of C4 exposes a highly reactive thioester bond on the C4b molecule that allows it to bind covalently to molecules in the immediate vicinity of its site of activation. In innate immunity, C4 cleavage is catalyzed by a C1 or MBL complex bound to the pathogen surface, and C4b 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 this hydrolysis reaction irreversibly inactivates C4b (Fig. 2.14). This helps to prevent C4b from diffusing from its site of activation on the microbial surface and becoming coupled to host cells.

C2 becomes susceptible to cleavage by C1s only when it is bound by C4b, and the C2b serine protease is thereby also confined to the pathogen surface, where it remains associated with C4b, forming a C3 convertase. The activation of C3 molecules thus also occurs at the surface of the pathogen. Furthermore, the C3b cleavage product is also rapidly inactivated unless it binds covalently by the same mechanism as C4b, and it therefore opsonizes only the surface on which complement activation has taken place.

2-9. Hydrolysis of C3 causes initiation of the alternative pathway of complement

The third pathway of complement activation is called the alternative pathway because it was discovered as a second, or ‘alternative,’ pathway for complement activation after the classical pathway had been defined. This pathway can proceed on many microbial surfaces in the absence of specific antibody, and it leads to the generation of a distinct C3 convertase designated C3b,Bb. In contrast to the classical and MB-lectin pathways of complement activation, the alternative pathway does not depend on a pathogen-binding protein for its initiation; instead it is initiated through the spontaneous hydrolysis of C3, as shown in the top three panels of Fig. 2.15. The distinctive components of the pathway are listed in Fig. 2.16. A number of mechanisms ensure that the activation pathway will only proceed on the surface of a pathogen.

C3 is abundant in plasma, and C3b is produced at a significant rate by spontaneous cleavage (also known as ‘tickover’). This occurs through the spontaneous hydrolysis of the thioester bond in C3 to form C3(H₂O) which has an altered conformation, allowing binding of the plasma protein factor B. The binding of B by C3(H₂O) then allows a plasma protease called factor D to cleave factor B to Ba and Bb, the latter remaining associated with C3(H₂O) to form the C3(H₂O)Bb complex. This complex is a fluid-phase C3 convertase, and although it is only formed in small amounts it can cleave many molecules of C3 to C3a and C3b. Much of this C3b is inactivated by hydrolysis, but some attaches covalently, through its reactive thioester group, to the surfaces of host cells or to pathogens. C3b bound in this way is able to bind factor B, allowing its cleavage by factor D to yield the small fragment Ba and the active protease Bb. This results in formation of the alternative pathway C3 convertase, C3b,Bb (see Fig. 2.15).

When C3b binds to host cells, a number of complement-regulatory proteins, present in the plasma and on host cell membranes combine to prevent complement activation from proceeding. These proteins interact with C3b and either prevent the convertase from forming, or promote its rapid dissociation (see Fig. 2.15). Thus, the complement receptor 1 (CR1) and a membrane-attached protein known as decay-accelerating factor (DAF or CD55) compete 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 its inactive derivative iC3b. This is achieved by a plasma protease, factor I, in conjunction with C3b-binding proteins that can act as cofactors, such as CR1 and membrane cofactor of proteolysis (MCP or CD46), another host cell membrane protein. Factor H is another complement-regulatory protein in plasma that binds C3b and, like CR1, it is able to compete with factor B and displace Bb from the convertase in addition to acting as a cofactor for factor I. Factor H binds preferentially to C3b bound to vertebrate cells as it has an affinity for the sialic acid residues present on these cells.

By contrast, because pathogen surfaces lack these regulatory proteins and sialic acid residues, the C3b,Bb convertase can form and persist. Indeed, this process may be favored by a positive regulatory factor, known as properdin or factor P, which binds to many microbial surfaces and stabilizes the convertase. Deficiencies in factor P are associated with a heightened susceptibility to infection with Neisseria species. Once formed, the C3b,Bb convertase rapidly cleaves yet more C3 to C3b, which can bind to the pathogen and either act as an opsonin or reinitiate the pathway to form another molecule of C3b,Bb convertase. Thus, the alternative pathway activates through an amplification loop that can proceed on the surface of a pathogen, but not on a host cell. This same amplification loop enables the alternative pathway to contribute to complement activation initially triggered through the classical or MB-lectin pathways (Fig. 2.17).

The C3 convertases resulting from activation of the classical and MB-lectin pathways (C4b,2b) and from the alternative pathway (C3b,Bb) are apparently distinct. However, understanding of the complement system is simplified somewhat by recognition of the close evolutionary relationships between the different complement proteins. Thus the complement zymogens, factor B and C2, are closely related proteins encoded by homologous genes located in tandem in the major 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. Only one component of the alternative pathway appears entirely unrelated to its functional equivalents in the classical and MB-lectin pathways; this is 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 spontaneous C3 cleavage, and safe for the host because factor D has no other substrate than factor B when bound to C3b. This means that factor D only finds its substrate at a very low level in plasma, and at pathogen surfaces where the alternative pathway of complement activation is allowed to proceed.

Comparison of the different pathways of complement activation illustrates the general principle that most of the immune effector mechanisms that can be activated in a nonclonal fashion as part of the early nonadaptive host response against infection have been harnessed during evolution to be used as effector mechanisms of adaptive immunity. It is almost certain that the adaptive response evolved by adding specific recognition to the original nonadaptive system. This is illustrated particularly clearly in the complement system, because here the components are defined, and the functional homologues can be seen to be evolutionarily related (Fig. 2.18).

2-10. Surface-bound C3 convertase deposits large numbers of C3b fragments on pathogen surfaces and generates C5 convertase activity

The formation of C3 convertases is the point at which the three pathways of complement activation converge, because both the classical pathway and MB-lectin pathway convertases C4b,2b, and the alternative pathway convertase C3b,Bb have the same activity, and they initiate the same subsequent events. They both 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^–1, and up to 1000 molecules of C3b can bind in the vicinity of a single active C3 convertase (see Fig. 2.11). Thus, the main effect of complement activation is to deposit large quantities of C3b on the surface of the infecting pathogen, where it forms a covalently bonded coat that, as we will see, can signal the ultimate destruction of the pathogen by phagocytes.

The next step in the cascade is the generation of the C5 convertases. In the classical and the MB-lectin pathways, a C5 convertase is formed by the binding of C3b to C4b,2b to yield C4b,2b,3b. By the same token, the C5 convertase of the alternative pathway is formed by the binding of C3b to the C3 convertase to form C3b₂,Bb. C5 is captured by these C5 convertase complexes through binding to an acceptor site on C3b, and is then rendered susceptible to cleavage by the serine protease activity of C2b or Bb. This reaction, which generates C5b and C5a, is much more limited than cleavage of C3, as C5 can be cleaved only when it binds to C3b that is part of the C5 convertase complex. Thus, complement activation by both the alternative, MB-lectin and classical 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 C5a (Fig. 2.19).

2-11. Phagocyte ingestion of complement-tagged pathogens 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.

The five known types of receptor for bound complement components are listed, with their functions and distributions, in Fig. 2.20. The best-characterized is the C3b receptor CR1 (CD35), which is expressed on both macrophages and polymorphonuclear leukocytes. 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. 2.21). C5a binds to another receptor expressed by macrophages, the C5a receptor, which has seven membrane-spanning domains. Receptors of this type couple with intracellular guanine-nucleotide-binding proteins called G proteins, and the C5a receptor signals in this way. 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. C3a, which has inflammatory activities similar to those of C5a, although it is a less potent chemoattractant, binds to its own specific receptor, the C3a receptor, which is homologous in structure to the C5a receptor.

Three other complement receptors—CR2 (also known as CD21), CR3 (CD11b:CD18), and CR4 (CD11c:CD18)—bind to inactivated forms of C3b that remain attached to the pathogen surface. Like several other key components of complement, C3b is subject to regulatory mechanisms and can be cleaved into derivatives that cannot form an active convertase. One of the inactive derivatives of C3b, known as iC3b (see Section 2-9) acts as an opsonin in its own right when bound by the complement receptors CR2 or CR3. Unlike the binding of iC3b to CR1, the binding of iC3b to CR3 is sufficient on its own to stimulate phagocytosis. A second breakdown product of C3b, called C3dg, binds only to 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 a given pathogen will receive a strongly augmented signal on binding this pathogen if it is also coated with C3dg. The activation of complement can therefore contribute to producing a strong antibody response (see Chapters 6 and 9). This example of how an innate humoral immune response can contribute to activating adaptive humoral immunity parallels the contribution made by the innate cellular response of macrophages and dendritic cells to the initiation of a T-cell response, which we will discuss later in this chapter.

The central role of opsonization by C3b and its inactive fragments in the destruction of extracellular pathogens can be seen in the effects of various complement deficiency diseases. Whereas individuals deficient in any of the late components of complement are relatively unaffected, individuals deficient in C3 or in molecules that catalyze C3b deposition show increased susceptibility to infection by a wide range of extracellular bacteria, as we will see in Chapter 11.

2-12. Small fragments of some complement proteins can initiate a local inflammatory response

The small complement fragments C3a, C4a, and C5a act on specific receptors (see Fig. 2.20) to produce local inflammatory responses. When produced in large amounts or injected systemically, they induce a generalized circulatory collapse, producing a shocklike syndrome similar to that seen in a systemic allergic reaction involving IgE antibodies (see Chapter 12). Such a reaction is termed anaphylactic shock and these small fragments of complement are therefore often referred to as anaphylotoxins. Of the three, C5a is the most stable and has the highest specific biological activity. All three induce smooth muscle contraction and increase vascular permeability, but C5a and C3a also act on the endothelial cells lining blood vessels to induce adhesion molecules. In addition, C3a and C5a can activate the mast cells that populate submucosal tissues to release mediators such as histamine and TNF-α that cause similar effects. The changes induced by C5a and C3a recruit antibody, complement, and phagocytic cells to the site of an infection (Fig. 2.22), 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.

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, as well as increasing 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. C5a and C3a signal through transmembrane receptors that activate G proteins; thus the action of C5a in attracting neutrophils and monocytes is analogous to that of chemokines, which also act via G proteins to control cell migration.

2-13. 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. 2.23) to form a membrane-attack complex. The reactions leading to the formation of this complex are shown schematically in Fig. 2.24. 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 cell membrane.

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. 2.19). In the next stages, shown in Fig. 2.24, C5b initiates the assembly of the later complement components and their insertion into the cell membrane. First, one molecule of C5b binds one molecule of C6, and the C5b,6 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 C5b,6,7 complex allows the hydrophobic domain of C8α-γ to insert into the lipid bilayer. Finally, C8α-γ induces the polymerization of 10 to 16 molecules of C9 into a pore-forming structure called the membrane-attack complex. The membrane-attack complex, shown schematically and by electron microscopy in Fig. 2.24, 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 100 Å, allowing the free passage of solutes and water across the lipid bilayer. The disruption of 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.

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 of these components in host defense seems to be quite limited. To date, 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 most 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 13, it might have a major role in immunopathology.

2-14. Complement control proteins regulate all three pathways of complement activation and protect the host from its destructive effects

Given the destructive effects of complement, and the way in which its activation is rapidly amplified through a triggered-enzyme cascade, it is not surprising that there are several mechanisms to prevent its uncontrolled activation. As we have seen, the effector molecules of complement are generated through the sequential activation of zymogens, which are present in plasma in an inactive form. The activation of these zymogens usually occurs on a pathogen surface, and the activated complement fragments produced in the ensuing cascade of reactions usually bind nearby or are rapidly inactivated by hydrolysis. These two features of complement activation act as safeguards against uncontrolled activation. Even so, all complement components are activated spontaneously at a low rate in plasma, and activated complement components will sometimes bind proteins on host cells. The potentially damaging consequences are prevented by a series of complement control proteins, summarized in Fig. 2.25, which regulate the complement cascade at different points. As we saw in discussing the alternative pathway of complement activation (see Section 2-9) many of these control proteins specifically protect host cells while allowing complement activation to proceed on pathogen surfaces. The complement control proteins therefore allow complement to distinguish self from nonself.

The reactions that regulate the complement cascade are shown in Fig. 2.26. The top two panels show how the activation of C1 is controlled by a plasma serine proteinase inhibitor or serpin, the C1 inhibitor (C1INH). C1INH binds the active enzyme C1r:C1s, and causes it to dissociate from C1q, which remains bound to the pathogen. 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 angioneurotic edema, in which chronic spontaneous complement activation leads to the production of excess cleaved fragments of C4 and C2. The small fragment of C2, C2a, is further cleaved into a peptide, the C2 kinin, which causes extensive swelling—the most dangerous being local swelling in the trachea, which can lead to suffocation. Bradykinin, which has similar actions to C2 kinin, is also produced in an uncontrolled fashion in this disease, as a result of the lack of inhibition of another plasma protease, kallikrein, which is activated by tissue damage and also regulated by C1INH. This disease is fully corrected by replacing C1INH. The large activated fragments of C4 and C2, which normally combine to form the C3 convertase, do not damage host cells in such patients because C4b is rapidly inactivated in plasma (see Fig. 2.14) and the convertase does not form. Furthermore, any convertase that accidentally forms on a host cell is inactivated by the mechanisms described below.

The thioester bond of activated C3 and C4 is extremely reactive and has no mechanism for distinguishing an acceptor hydroxyl or amine group on a host cell from a similar group on the surface of a pathogen. A series of protective mechanisms, mediated by other proteins, has evolved to ensure that the binding of a small number of C3 or C4 molecules to host cell membranes results in minimal formation of C3 convertase and little amplification of complement activation. We have already encountered most of these mechanisms in the description of the alternative pathway (see Fig. 2.15), but we will consider them again here, as they are important regulators of the classical pathway convertase as well (see Fig. 2.26, second and third rows). The mechanisms can be divided into three categories. The first catalyze the cleavage of any C3b or C4b that does bind to host cells into inactive products. The complement-regulatory enzyme responsible is the plasma serine protease factor I; it circulates in active form but can only cleave C3b and C4b when they are bound to a cofactor protein. 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. There are two cell-membrane proteins that bind C3b and C4b and possess cofactor activity for factor I; these are CR1 and MCP (see Section 2-9). Microbial cell walls lack these protective proteins and cannot promote the breakdown of C3b and C4b. Instead, these proteins act as binding sites for factor B and C2, promoting complement activation. The importance of factor I can be seen in people with genetic 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.

There are also plasma proteins with cofactor activity for factor I. C4b is bound by a cofactor known as the C4b-binding protein (C4BP), which mainly acts as a regulator of the classical pathway in the fluid phase. C3b is bound in both the fluid phase and at cell membranes by a cofactor protein called factor H (see Section 2-9). Factor H is an important complement regulator at cell membranes, and at first sight it is not obvious how factor H can distinguish C3b bound to host cells or to a pathogen. However, the carbohydrate content of the cell membranes of bacterial pathogens differs from that of their hosts and this is the basis for the protective effect of factor H. Factor H has affinity for the terminal sialic acids of host cell membrane glycoproteins and this increases the binding of factor H to any C3b deposited on host cells. In contrast, factor H has a much lower affinity for C3b deposited on the cell walls of many bacteria, and factor B binds in preference, resulting in amplification of complement activation on bacterial cell surfaces. In effect, factor H and factor B compete for binding to C3b bound to cells. If factor B ‘wins,’ as is typically the case on a pathogen surface, then more C3b,Bb C3 convertase forms and complement activation is amplified. If factor H ‘wins,’ as is the case on cells of the host, then the bound C3b is catabolized by factor I to iC3b and C3dg and complement activation is inhibited.

The competition between factor H and factor B for binding to surface-bound C3b is an example of the second mechanism for inhibiting complement activation on host cell membranes. A number of proteins competitively inhibit the binding of C2 to cell-bound C4b and of factor B to cell-bound C3b, thereby inhibiting convertase formation. These proteins bind to C3b and C4b on the cell surface, and also mediate protection against complement through the third mechanism, which is to augment the dissociation of C4b,2b and C3b,Bb convertases that have already formed. Host cell membrane molecules that regulate complement through both these mechanisms include DAF (see Section 2-9) and CR1, which promotes dissociation of convertase in addition to its 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), complement control protein (CCP) repeat, or (especially in Japan) the sushi domain.

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 into membranes. We saw in Section 2-13 that the membrane-attack complex polymerizes onto C5b molecules released from C5 convertase. This 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 bind to the C5b,6,7 complex and thereby inhibit its random insertion into cell membranes. The most important is probably C8β itself, when it binds to C5b,6,7 in the fluid phase. Host cell membranes also contain an intrinsic protein, CD59 or protectin, which inhibits the binding of C9 to the C5b,6,7,8 complex (see Fig. 2.26, bottom row). CD59 and DAF are both linked to the cell surface by a phosphoinositol glycolipid (PIG) tail, like many other membrane proteins. One of the enzymes involved in the synthesis of PIG tails is encoded on chromosome X. 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 CD59 only are also susceptible to destruction as a result of spontaneous activation of the complement cascade.

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 reactions that occurs on the surface of pathogens and generates active components with various effector functions. There are three pathways of complement activation: the classical pathway, which is triggered directly by pathogen or indirectly by antibody binding to the pathogen surface; the MB-lectin pathway; and the alternative pathway, which also provides an amplification loop for the other two pathways. All three pathways can be initiated independently of antibody as part of innate immunity. 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 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, C4, and especially C5, recruit phagocytes to sites of infection and activate them by binding to specific trimeric G protein-coupled receptors. 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, binding C5 to make it susceptible to cleavage by C2b or Bb. The larger C5b fragment triggers the assembly of a membrane-attack complex, which can result in the lysis of certain pathogens. The activity of complement components is modulated by a system of regulatory proteins that prevent tissue damage as a result of inadvertent binding of activated complement components to host cells or spontaneous activation of complement components in plasma.

2-15. Receptors with specificity for pathogen surfaces recognize patterns of repeating structural motifs

The surfaces of microorganisms typically bear repeating patterns of molecular structure. The innate immune system recognizes such pathogens by means of receptors that bind features of these regular patterns; these receptors are sometimes known as pattern-recognition molecules. The mannan-binding lectin that initiates the MB-lectin pathway of complement activation (see Section 2-5) is one such receptor, as shown by structural studies of its binding. As illustrated in Fig. 2.28, pathogen recognition and discrimination from self is due to recognition of a particular orientation of certain sugar residues, as well as their spacing, which is found only on pathogenic microbes and not on host cells. Other members of the collectin family also bind pathogens directly and function in innate immunity. As we saw in Section 2-6, the collectin C1q is able to bind directly to pathogen surfaces and initiate complement activation through the classical pathway. In addition, other collectins are made in the liver as part of the acute-phase response, which will be described in the last part of the chapter. The exact structures recognized by these other collectins have not yet been defined, but all collectins have multiple carbohydrate-recognition domains attached to a collagen helix and are thought to bind pathogen surfaces in a similar way to mannan-binding lectin.

The interaction of these soluble receptors with pathogens leads in turn to binding of the receptor:pathogen complex by phagocytes, either through direct interaction with the pathogen-binding receptor, or through receptors for complement, thus promoting phagocytosis and killing of the bound pathogen (see Section 2-3) and the induction of other cellular responses.

Phagocytes are also equipped with several cell-surface receptors that recognize pathogen surfaces directly. Among these are the macrophage mannose receptor (see Section 2-3). This receptor is a cell-bound C-type lectin that binds certain sugar molecules found on the surface of many bacteria and some viruses, including the human immunodeficiency virus (HIV). Its recognition properties are very similar to those of mannan-binding lectin (see Fig. 2.28) and, like mannan-binding lectin, it is a multipronged molecule with several carbohydrate-recognition domains. Because it is a transmembrane cell-surface receptor, however, it can function directly as a phagocytic receptor. A second set of phagocytic receptors, called scavenger receptors, recognize certain anionic polymers and also acetylated low-density lipoproteins. These receptors are a structurally heterogeneous set of molecules, existing in at least six distinct molecular forms. Some scavenger receptors recognize structures that are shielded by sialic acid on normal host cells. These receptors are involved in the removal of old red blood cells that have lost sialic acid, as well as in the recognition and removal of pathogens. There are other recognition targets, many of which still need to be characterized.

2-16. Receptors on phagocytes can signal the presence of pathogens

In addition to triggering phagocytosis, binding of pathogens by macrophages can also trigger the induced responses of innate immunity, and responses that eventually lead to the induction of adaptive immunity. Not all the receptors on phagocytes appear to transmit such signals. The best-defined activation pathway of this type is triggered through a family of evolutionarily conserved transmembrane receptors that appear to function exclusively as signaling receptors. These receptors, known as the Toll receptors, were first described in the fruit-fly. They appear not to recognize and bind pathogens directly, but clearly are involved in signaling the appropriate response to different classes of pathogen. In the fruit-fly, the Toll receptor itself triggers the production of antifungal peptides in response to fungal infections, while a different member of the Toll family is involved in activating an antibacterial response. In mammals, a Toll-family protein, called Toll-like receptor 4, or TLR-4, signals the presence of LPS by associating with CD14, the macrophage receptor for LPS. TLR-4 is also involved in the immune response to at least one virus, respiratory syncytial virus, although in this case the nature of the stimulating ligand is not known. Another mammalian Toll-like receptor, TLR-2, signals the presence of a different set of microbial constituents, which include the proteoglycans of gram-positive bacteria, although how it recognizes these is not known. TLR-4 and TLR-2 induce similar but distinct signals, as shown by the distinct responses resulting from LPS signaling through TLR-4 and proteoglycan signaling through TLR-2. There are at least nine distinct proteins in this newly discovered family in mammals, and further functions of Toll-like receptors may soon be revealed as mice lacking one or other of these proteins are produced and analyzed.

2-17. The effects of bacterial lipopolysaccharide on macrophages are mediated by CD14 binding to Toll-like receptor 4

Bacterial LPS is a cell-wall component of gram-negative bacteria that has long been known for its ability to induce a dramatic systemic reaction known as septic shock. Perhaps because of this, the best-characterized proteins in innate immunity are the plasma protein LPS-binding protein (LBP), and the receptor protein CD14 (see Section 2-3), which binds LBP-bound LPS. Both LBP and CD14 have leucine-rich repeat motifs. Although the structural details of the binding are not yet characterized, the LPS:LBP complex binds to CD14, which is either free in the plasma or bound to the cell surface by a phosphoinositol glycolipid tail (Fig. 2.29, top two panels). This binding triggers a cell response, but until recently the mechanism by which the signals were transduced across the cell membrane was unknown.

Mice were discovered that were genetically unresponsive to LPS and did not suffer from septic shock, but had no defects in LBP and CD14. The gene responsible was tracked down by positional cloning and turned out to be the gene for TLR-4, which had suffered inactivating mutations in these mice. The response to LPS could be restored by inserting a transgene encoding TLR-4 into the mouse germline, proving that the defect in TLR-4 was entirely responsible for the loss of responsiveness to LPS. It was subsequently found that TLR-4 binds to the CD14:LBP:LPS complex through a leucine-rich region in CD14’s extracellular domain.

LPS responsiveness is most commonly assessed experimentally by the ability to induce LPS-mediated septic shock. This syndrome is the result of overwhelming secretion of cytokines, particularly of TNF-α, often as a result of an uncontrolled systemic bacterial infection. We will discuss the pathogenesis of septic shock later in this chapter, and will see that it is an undesirable consequence of the same effector actions of TNF-α that are important in containing local infections. The benefits of TLR-4 signaling are clearly illustrated by the mutant mice that lack TLR-4 function; although resistant to septic shock, they are highly sensitive to LPS-bearing pathogens such as Salmonella typhimurium, a natural pathogen of mice. Some people with gram-negative sepsis, an uncontrolled infection of the bloodstream with gram-negative bacteria, which indicates failure to contain an infection locally, have mutations in the TLR-4 gene, showing that TLR-4 is important in protecting against gram-negative sepsis in humans. Only a small fraction of patients have such mutations, however, so there may be other specific defects that contribute to a failure to respond adequately to gram-negative bacteria.

When TLR-4 binds to CD14 complexed with its LBP:LPS ligand, TLR-4 sends a signal to the nucleus that activates the transcription factor NFκB (Fig. 2.29, bottom panel). We will describe the signaling pathway used by TLR-4 in detail in Chapter 6. This signaling pathway was first discovered as the pathway used by the Toll receptor to determine dorsoventral body pattern during embryogenesis in the fruit-fly Drosophila, and we will therefore call it the Toll pathway. The pathway was recently shown to participate in defense against infection in adult flies, and a similar pathway is used by plants in their defense against viruses. Thus the Toll pathway is an ancient signaling pathway that appears to be used in innate immune defense in most or all multicellular organisms.

2-18. Activation of Toll-like receptors triggers the production of pro-inflammatory cytokines and chemokines, and the expression of co-stimulatory molecules

The Toll signaling pathway in adult flies induces the production of several antimicrobial peptides that contribute to the fly’s defense against infection. In humans and all other vertebrates examined, activation of NFκB by the Toll pathway leads to the production of several important mediators of innate immunity, such as cytokines and chemokines. At the same time, Toll signaling induces molecules that are essential for the induction of adaptive immune responses. These are known as co-stimulatory molecules, and they will be considered in detail in Chapter 8. The co-stimulatory molecules, called B7.1 (CD80) and B7.2 (CD86), are cell-surface proteins that are expressed by both macrophages and tissue dendritic cells in response to LPS signaling through TLR-4. It is the presence of these molecules along with the microbial antigens presented by macrophages and dendritic cells (see Section 1-6) that activates the CD4 T cells required to initiate most adaptive immune responses. To encounter a CD4 T cell, the antigen-presenting dendritic cell must migrate to a nearby lymph node through which circulating T cells pass, and this migration is stimulated by cytokines such as TNF-α, which are also induced through signaling by TLR-4. Thus, the activation of adaptive immunity depends on molecules induced as a consequence of innate immune recognition and signaling (Fig. 2.30).

Substances such as LPS that induce co-stimulatory activity have been used for years in mixtures that are co-injected with protein antigens to enhance their immunogenicity. These substances are known as adjuvants (see Appendix I, Section A-4), and it was found empirically that the best adjuvants contained microbial components. Pathogen components that can induce macrophages and tissue dendritic cells to express co-stimulatory molecules and cytokines include glycans, mannans, and the mycobacterial extract muramyl dipeptide. The receptor TLR-2 has recently been shown to be essential for the response to proteoglycans and it is thought that all these pathogen components are recognized by pattern-recognition molecules that then signal, often through Toll-like receptors, to induce co-stimulatory molecules and cytokines. The exact profile of cytokines produced varies according to the receptors involved and, as we will see in Chapters 8 and 10, this will in turn influence the functional character of the adaptive immune response that develops in their presence. In this way, the ability of the innate immune system to discriminate between different types of pathogen is used to ensure an appropriate type of adaptive immune response.

Summary

The innate immune system uses a diversity of receptors to recognize and respond to pathogens. Those that recognize pathogen surfaces directly often bind to repeating patterns, for example, of carbohydrate or lipid moieties, that are characteristic of microbial surfaces but are not found on host cells. Some of these receptors, for example, the macrophage mannose receptor, directly stimulate phagocytosis, while others are produced as secreted molecules that promote the phagocytosis of pathogens by opsonization or by the activation of complement. The innate immune system receptors that recognize pathogens also have an important role in signaling for the induced responses responsible for local inflammation, the recruitment of new effector cells, the containment of local infection, and the initiation of an adaptive immune response. Such signals can be transmitted through a family of signaling receptors, known as the Toll-like receptors, that have been highly conserved across species and that serve to activate host defense through a signaling pathway that operates in most multicellular organisms. In mammals, Toll-like receptors also play a key role in enabling the initiation of adaptive immunity. TLR-4 detects the presence of gram-negative bacteria through its association with the peripheral membrane protein CD14, which is a receptor for bacterial LPS bound to its binding protein, LBP. TLR-2 signals in response to microbial proteoglycans, although how it recognizes them is not yet known. Ligation of TLR-2 or TLR-4 activates an evolutionarily ancient signaling pathway that leads to the activation of the transcription factor NFκB, and the induction of a variety of genes, including genes for cytokines, chemokines, and co-stimulatory molecules that play essential roles in directing the course of the adaptive immune response later in infection.

2-19. Activated macrophages secrete a range of cytokines that have a variety of local and distant effects

Cytokines are small proteins (~25 kDa) that are released by various cells in the body, usually in response to an activating stimulus, and induce responses through binding to specific receptors. They can act in an autocrine manner, affecting the behavior of the cell that releases the cytokine, or in a paracrine manner, affecting the behavior of adjacent cells. Some cytokines can act in an endocrine manner, affecting the behavior of distant cells, although this depends on their ability to enter the circulation and on their half-life. Chemokines are a class of cytokines that have chemoattractant properties, inducing cells with the appropriate receptors to migrate toward the source of the chemokine. The cytokines secreted by macrophages in response to pathogens are a structurally diverse group of molecules and include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), TNF-α, and the chemokine interleukin-8 (IL-8). The name interleukin (IL) followed by a number (for example IL-1, IL-2, and so on) was coined in an attempt to develop a standardized nomenclature for molecules secreted by, and acting on, leukocytes. However, this became confusing when an ever-increasing number of cytokines with diverse origins, structures, and effects were discovered, and although the IL designation is still used, it is hoped that eventually a nomenclature based on cytokine structure will be developed. The cytokines and their receptors are grouped according to their structure in the appendices at the end of this book (cytokines are listed in Appendix III and chemokines in Appendix IV). There are three major structural families: the hematopoietin family, which includes growth hormones as well as many interleukins with roles in both adaptive and innate immunity; the TNF family, which functions in both innate and adaptive immunity and includes some membrane-bound members; and the chemokine family, which we discuss below. Of the macrophage-derived interleukins shown in Fig. 2.31, IL-6 belongs to the large family of hematopoietins, TNF-α is obviously part of the TNF family, while IL-1 and IL-12 are structurally distinct. All have important local and systemic effects that contribute to both innate and adaptive immunity, and these are summarized in Fig. 2.31.

The recognition of different classes of pathogen may involve signaling through distinct receptors and result in some variation in the cytokines induced. The study of this is still in its infancy, but it is thought to be a way in which appropriate responses can be selectively activated as the released cytokines orchestrate the next phase of host defense. We will see how TNF-α, which is elicited by LPS-bearing pathogens, is particularly important in containing infection by these pathogens, and, how the release of different chemokines can recruit and activate different types of effector cells.

2-20. Chemokines released by phagocytes recruit cells to sites of infection

Among the cytokines released in infected tissue in the earliest phases of infection are members of a family of chemoattractant cytokines known as chemokines. These molecules induce directed chemotaxis in nearby responsive cells and were discovered relatively recently. Because they were first detected in cytokine assays, they were initially named as interleukins: interleukin-8 (IL-8) was the first chemokine to be cloned and characterized, and it remains typical of this family (Fig. 2.32, top). All the chemokines are related in amino acid sequence and their receptors are all integral membrane proteins containing seven membrane-spanning helices. This structure is characteristic of receptors such as rhodopsin (Fig. 2.32, bottom) and the muscarinic acetylcholine receptor, which are coupled to G proteins; the chemokine receptors also signal through coupled G proteins. Chemokines function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils, and other effector cells from the blood to sites of infection. They can be released by many different types of cell and serve to guide cells involved in innate immunity and also the lymphocytes in adaptive immunity, as we will learn in Chapters 8–10. Some chemokines also function in lymphocyte development, migration, and angiogenesis (the growth of new blood vessels). The properties of a variety of chemokines are listed in Fig. 2.33; quite why there are so many chemokines is not yet known, and neither is the exact role of each in defense against infection.

Members of the chemokine family fall mostly into two broad groups—CC chemokines with two adjacent cysteines near the amino terminus, and CXC chemokines, in which the equivalent two cysteine residues are separated by another amino acid. The two groups of chemokines act on different sets of receptors. CC chemokines bind to CC chemokine receptors, of which there are nine so far, designated CCR1–9. CXC chemokines bind to CXC receptors; there are five of these, CXCR1–5. These receptors are expressed on different cell types; in general, CXC chemokines with an Glu-Leu-Arg (ELR) tripeptide motif immediately before the first cysteine promote the migration of neutrophils. IL-8 is an example of this type of chemokine. Other CXC chemokines that lack this motif, such as the B-lymphocyte chemokine (BLC), guide lymphocytes to their proper destination. The CC chemokines promote the migration of monocytes or other cell types. An example is macrophage chemoattractant protein-1 (MCP-1). IL-8 and MCP-1 have similar, although complementary, functions: IL-8 induces neutrophils to leave the bloodstream and migrate into the surrounding tissues; MCP-1, in contrast, acts on monocytes, inducing their migration from the bloodstream to become tissue macrophages. Other CC chemokines such as RANTES may promote the infiltration into tissues of a range of leukocytes including effector T cells (see Section 10-8), with individual chemokines acting on different subsets of cells. The only known C chemokine (with only one cysteine) is called lymphotactin and is thought to attract T-cell precursors to the thymus. A newly discovered molecule called fractalkine is unusual in several ways: it has three amino acid residues between the two cysteines, making it a CX₃C chemokine; it is multimodular; and it is tethered to the membrane of the cells that express it, where it serves both as a chemoattractant and as an adhesion protein. We will return to the discussion of chemokines in Chapter 10.

The role of chemokines such as IL-8 and MCP-1 in cell recruitment is twofold. First, they act on the leukocyte as it rolls along endothelial cells at sites of inflammation, converting this rolling into stable binding by triggering a change of conformation in the adhesion molecules known as leukocyte integrins. This allows the leukocyte to cross the blood vessel wall by squeezing between the endothelial cells, as we will see when we describe the process of extravasation. Second, the chemokines direct the migration of the leukocyte along a gradient of the chemokine that increases in concentration toward the site of infection. This is achieved by the binding of the small, soluble chemokines to proteoglycan molecules in the extracellular matrix and on endothelial cell surfaces, thus displaying the chemokines on a solid substrate along which the leukocytes can migrate.

Chemokines can be produced by a wide variety of cell types in response to bacterial products, viruses, and agents that cause physical damage, such as silica or the urate crystals that occur in gout. Thus, infection or physical damage to tissues sets in motion the production of chemokine gradients that can direct phagocytes to sites where they are needed. In addition, peptides that act as chemoattractants for neutrophils are made by bacteria themselves. All bacteria produce proteins with an amino-terminal N-formylated methionine, and, as discovered many years ago, the f-Met-Leu-Phe (fMLP) peptide is a potent chemotactic factor for inflammatory cells, especially neutrophils. The fMLP receptor is also a G protein-coupled receptor like the receptors for chemokines and for the complement fragments C5a, C3a, and C4a. Thus, there is a common mechanism for attracting neutrophils, whether by complement, chemokines, or bacterial peptides. Neutrophils are the first to arrive in large numbers at a site of infection, with monocytes and immature dendritic cells being recruited later. The complement peptide C5a, and the chemokines IL-8 and MCP-1 also activate their respective target cells, so that not only are neutrophils and macrophages brought to potential sites of infection but, in the process, they are armed to deal with any pathogens they may encounter. In particular, neutrophils exposed to IL-8 and the cytokine TNF-α (see Fig. 2.37, and Section 2-23) are activated to produce the respiratory burst that generates oxygen radicals and nitric oxide, and to release their stored lysosomal contents, thus contributing both to host defense and to the tissue destruction and pus formation seen in local sites of infection with pyogenic bacteria.

Chemokines do not act alone in cell recruitment, which also requires the action of vasoactive mediators to bring leukocytes close to the blood vessel endothelium (see Section 2-4) and cytokines such as TNF-α to induce the necessary adhesion molecules on the endothelial cells. We will now turn to the molecules that mediate leukocyte–endothelium adhesion, and then describe the process of leukocyte extravasation step by step, as it is known to occur for neutrophils and monocytes.

2-21. Cell-adhesion molecules control interactions between leukocytes and endothelial cells during an inflammatory response

The recruitment of activated phagocytes to sites of infection is one of the most important functions of innate immunity. Recruitment occurs as part of the inflammatory response and is mediated by cell-adhesion molecules that are induced on the surface of the local blood vessel endothelium. Before we consider the process of inflammatory cell recruitment we will first describe some of the cell-adhesion molecules involved.

A significant barrier to understanding cell-adhesion molecules is their nomenclature. Most cell-adhesion molecules, especially those on leukocytes, which are relatively easy to analyze functionally, were named after the effects of specific monoclonal antibodies against them, and were only later characterized by gene cloning. Their names therefore bear no relation to their structure; for instance, the leukocyte functional antigens, LFA-1, LFA-2, and LFA-3, are actually members of two different protein families. In Fig. 2.34, the adhesion molecules are grouped according to their molecular structure, which is shown in schematic form, alongside their different names, sites of expression, and ligands. Three families of adhesion molecules are important for leukocyte recruitment. The selectins are membrane glycoproteins with a distal lectinlike domain that binds specific carbohydrate groups. Members of this family are induced on activated endothelium and initiate endothelial– leukocyte interactions by binding to fucosylated oligosaccharide ligands on passing leukocytes (see Fig. 8.5). The next step in leukocyte recruitment depends on tighter adhesion, which is due to intercellular adhesion molecules (ICAMs) on the endothelium binding to heterodimeric proteins of the integrin family on leukocytes. We have already encountered two of the leukocyte integrins that function as complement receptors (CR3 and CR4). The leukocyte integrins important for extravasation are LFA-1 (α_L:β₂) and Mac-1 (α_M:β₂; another name for CR3) and they bind to both ICAM-1 and ICAM-2 (Fig. 2.35). Strong adhesion between leukocytes and endothelial cells is promoted by the induction of ICAM-1 on inflamed endothelium and the activation of a conformational change in LFA-1 and Mac-1 in response to chemokines. The importance of the leukocyte integrins in inflammatory cell recruitment is illustrated by the disease leukocyte adhesion deficiency, which stems from a defect in the β₂ chain common to both LFA-1 and Mac-1. People with this disease suffer from recurrent bacterial infections and impaired healing of wounds.

The activation of endothelium is driven by interactions with macrophage cytokines, particularly TNF-α, which induces rapid externalization of granules in the endothelial cells called Weibel–Palade bodies. These granules contain preformed P-selectin, which is thus expressed within minutes on the surface of local endothelial cells following production of TNF-α by macrophages. The same effect can be produced directly by exposing cultured human umbilical vein epithelial cells (HUVEC) to LPS, demonstrating that HUVEC can directly sense the presence of infection. Shortly after the appearance of P-selectin on the cell surface, mRNA encoding E-selectin is synthesized, and within 2 hours, the endothelial cells are mainly expressing E-selectin. Both these proteins interact with sulfated-sialyl-Lewis^x, which is present on the surface of neutrophils.

Resting endothelium carries low levels of ICAM-2, apparently in all vascular beds. This may be used by circulating monocytes to navigate out of the vessels and into their tissue sites, which happens continuously and essentially ubiquitously. However, upon exposure to TNF-α, local expression of ICAM-1 is strongly induced on the endothelium of small vessels within the infectious focus. This, in turn, binds to LFA-1 on circulating monocytes and polymorpho-nuclear leukocytes, in particular neutrophils, as shown in Fig. 2.35.

Cell-adhesion molecules have many other roles in the body, directing many aspects of tissue and organ development. In this brief description, we have considered only those that participate in the recruitment of inflammatory cells in the hours to days after the establishment of infection.

2-22. Neutrophils make up the first wave of cells that cross the blood vessel wall to enter inflammatory sites

The physical changes that accompany the initiation of the inflammatory response have been described in Section 2-4; here we give a step-by-step account of how the required effector cells are recruited into sites of infection. Under normal conditions, leukocytes are restricted to the center of small blood vessels, where the flow is fastest. In inflammatory sites, where the vessels are dilated, the slower blood flow allows the leukocytes to move out of the center of the blood vessel and interact with the vascular endothelium. Even in the absence of infection, monocytes migrate continuously into the tissues, where they differentiate into macrophages; during an inflammatory response, the induction of adhesion molecules on the endothelial cells, as well as induced changes in the adhesion molecules expressed on leukocytes, recruit large numbers of circulating leukocytes, initially neutrophils and later monocytes, into the site of an infection. The migration of leukocytes out of blood vessels, a process known as extravasation, is thought to occur in four steps. We will describe this process as it is known to occur for monocytes and neutrophils (Fig. 2.36).

The first step involves selectins. P-selectin, which is carried inside endothelial cells in Weibel–Palade bodies, appears on endothelial cell surfaces within a few minutes of exposure to leukotriene B4, the complement fragment C5a, or histamine, which is released from mast cells in response to C5a. The appearance of P-selectin can also be induced by exposure to TNF-α or LPS, and both of these have the additional effect of inducing the synthesis of a second selectin, E-selectin, which appears on the endothelial cell surface a few hours later. These selectins recognize the sulfated-sialyl-Lewis^x moiety of certain leukocyte glycoproteins that are exposed on the tips of microvilli. The interaction of P-selectin and E-selectin with these glycoproteins allows monocytes and neutrophils to adhere reversibly to the vessel wall, so that circulating leukocytes can be seen to ‘roll’ along endothelium that has been treated with inflammatory cytokines (see Fig. 2.36, top panel). This adhesive interaction permits the stronger interactions of the next step in leukocyte migration.

This second step depends upon interactions between the leukocyte integrins LFA-1 and Mac-1 with molecules on endothelium such as ICAM-1, which is also induced on endothelial cells by TNF-α (see Fig. 2.36, bottom panel). LFA-1 and Mac-1 normally adhere only weakly, but IL-8 or other chemokines, bound to proteoglycans on the surface of endothelial cells, trigger a conformational change in LFA-1 and Mac-1 on the rolling leukocyte, which greatly increases its adhesive properties. In consequence, the leukocyte attaches firmly to the endothelium and rolling is arrested.

In the third step, the leukocyte extravasates, or crosses the endothelial wall. This step also involves LFA-1 and Mac-1, as well as a further adhesive interaction involving an immunoglobulin-related molecule called PECAM or CD31, which is expressed both on the leukocyte and at the intercellular junctions of endothelial cells. These interactions enable the phagocyte to squeeze between the endothelial cells. It then penetrates the basement membrane (an extracellular matrix structure) with the aid of proteolytic enzymes that break down the proteins of the basement membrane. The movement through the vessel wall is known as diapedesis, and enables phagocytes to enter the subepithelial tissues.

The fourth and final step in extravasation is the migration of leukocytes through the tissues under the influence of chemokines. As discussed in Section 2-20, chemokines such as IL-8 are produced at the site of infection and bind to proteoglycans in the extracellular matrix. They form a matrix-associated concentration gradient along which the leukocyte can migrate to the focus of infection. IL-8 is released by the macrophages that first encounter pathogens and recruits neutrophils, which enter the infected tissue in large numbers in the early part of the induced response. Their influx usually peaks within the first six hours of an inflammatory response, while monocytes can be recruited later, through the action of chemokines such as MCP-1. Once in an inflammatory site, neutrophils are able to eliminate many pathogens by phagocytosis. They act as phagocytic effectors in an innate immune response through receptors for complement and other opsonizing proteins of the innate immune system as well as by recognizing pathogens directly. In addition, as we will see in Chapter 9, they act as phagocytic effectors in humoral adaptive immunity. The importance of neutrophils is dramatically illustrated by diseases or treatments that severely reduce neutrophil numbers. Such patients are said to have neutropenia, and are very susceptible to infection with numerous pathogens. Restoring neutrophil levels in such patients by transfusion of neutrophil-rich blood fractions or by stimulating their production with specific growth factors largely corrects this susceptibility.

2-23. Tumor necrosis factor-α is an important cytokine that triggers local containment of infection, but induces shock when released systemically

Inflammatory mediators also stimulate endothelial cells to express proteins that trigger blood clotting in the local small vessels, occluding them and cutting off blood flow. This can be important in preventing the pathogen from entering the bloodstream and spreading through the blood to organs all over the body. Instead, the fluid that has leaked into the tissue in the early phases of carries the pathogen enclosed in phagocytic cells, especially dendritic cells, via the lymph to the regional lymph nodes, where an adaptive immune response can be initiated. The importance of TNF-α in the containment of local infection is illustrated by experiments in which rabbits are infected locally with a bacterium. Normally, the infection will be contained at the site of the inoculation; if, however, an injection of anti-TNF-α antibody is also given to block the action of TNF-α, the infection spreads via the blood to other organs.

Once an infection spreads to the bloodstream, however, the same mechanisms by which TNF-α so effectively contains local infection instead become catastrophic (Fig. 2.37). The presence of infection in the bloodstream, known as sepsis, is accompanied by the release of TNF-α by macrophages in the liver, spleen, and other sites. The systemic release of TNF-α causes vasodilation and loss of plasma volume owing to increased vascular permeability, leading to shock. In septic shock, disseminated intravascular coagulation (blood clotting) is also triggered by TNF-α, leading to the generation of clots in many small vessels and the massive consumption of clotting proteins, thus causing the patient’s ability to clot blood appropriately to be lost. This condition frequently leads to the failure of vital organs such as the kidneys, liver, heart, and lungs, which are quickly compromised by the failure of normal perfusion; consequently, septic shock has a high mortality rate.

Mice with a mutant TNF-α receptor gene are resistant to septic shock; however, such mice are also unable to control local infection. Although the features of TNF-α that make it so valuable in containing local infection are precisely those that give it a central role in the pathogenesis of septic shock, it is clear from the evolutionary conservation of TNF-α that its benefits in the former area far outweigh the devastating consequences of its systemic release.

2-24. Cytokines released by phagocytes activate the acute-phase response

As well as their important local effects, the cytokines produced by macrophages have long-range effects that contribute to host defense. One of these is the elevation of body temperature, which is mainly caused by TNF-α, IL-1, and IL-6. These are termed endogenous pyrogens because they cause fever and derive from an endogenous source rather than from bacterial components. Fever is generally beneficial to host defense; most pathogens grow better at lower temperatures and adaptive immune responses are more intense at elevated temperatures. Host cells are also protected from the deleterious effects of TNF-α at raised temperatures.

The effects of TNF-α, IL-1, and IL-6 are summarized in Fig. 2.38. One of the most important of these is the initiation of a response known as the acute-phase response (Fig. 2.39). This involves a shift in the proteins secreted by the liver into the blood plasma and results from the action of IL-1, IL-6, and TNF-α on hepatocytes. In the acute-phase response, levels of some plasma proteins go down, while levels of others increase markedly. The proteins whose synthesis is induced by TNF-α, IL-1, and IL-6 are called acute-phase proteins. Several of these are of particular interest because they mimic the action of antibodies, but, unlike antibodies, these proteins have broad specificity for pathogen-associated molecular patterns and depend only on the presence of cytokines for their production.

One of these proteins, C-reactive protein, is a member of the pentraxin protein family, so called because they are formed from five identical subunits. C-reactive protein is another example of a multipronged pathogen-recognition molecule, and binds to the phosphorylcholine portion of certain bacterial and fungal cell-wall lipopolysaccharides. Phosphorylcholine is also found in mammalian cell membrane phospholipids but in a form that cannot bind to C-reactive protein. When C-reactive protein binds to a bacterium, it is not only able to opsonize it but can also activate the complement cascade by binding to C1q, the first component of the classical pathway of complement activation. The interaction with C1q involves the collagen-like parts of C1q, rather than the globular heads contacted by antibody and pathogen surfaces, but the same cascade of reactions is initiated.

The second acute-phase protein of interest is mannan-binding lectin, which we have already met as a pathogen-binding molecule (see Fig. 2.13) and as a trigger for the complement cascade (see Section 2-7). Mannan-binding lectin is found in normal serum at low levels but is produced in increased amounts during the acute-phase response. It acts as an opsonin for monocytes, which, unlike tissue macrophages, do not express the macrophage mannose receptor. Two other proteins with opsonizing properties that are produced by the liver in increased quantities during the acute-phase response are the pulmonary surfactants A and D. Like mannan-binding lectin and C1q these are members of the collectin family, binding to pathogen surfaces through globular lectin-domains attached to a collagen-like stalk. Pulmonary surfactants A and D are found along with macrophages in the alveolar fluid of the lung and are important in promoting the phagocytosis of respiratory pathogens such as Pneumocystis carinii, one of the main causes of pneumonia in patients with AIDS.

Thus, within a day or two, the acute-phase response provides the host with several proteins with the functional properties of antibodies that bind a broad range of pathogens. However, unlike antibodies, they have no structural diversity and are made in response to any stimulus that triggers the release of TNF-α, IL-1, and IL-6, so their synthesis is not specifically induced and targeted.

A final distant effect of the cytokines produced by phagocytes is to induce a leukocytosis, an increase in circulating neutrophils. The neutrophils come from two sources: the bone marrow, from which mature leukocytes are released in increased numbers; and sites in blood vessels where they are attached loosely to endothelial cells. Thus, the effects of these cytokines contribute to the control of infection while the adaptive immune response is being developed. As shown in Fig. 2.30, TNF-α also has a role in this, as it stimulates the migration of dendritic cells from their sites in peripheral tissues to the lymph node, and their maturation into nonphagocytic but highly co-stimulatory antigen-presenting cells.

2-25. Interferons induced by viral infection make several contributions to host defense

Infection of cells with viruses induces the production of proteins that are known as interferons because they were found to interfere with viral replication in previously uninfected tissue culture cells. They are believed to have a similar role in vivo, blocking the spread of viruses to uninfected cells. These antiviral effector molecules, called interferon-α (IFN-α) and interferon-β (IFN-β), are quite distinct from interferon-γ (IFN-γ). This is not directly induced by viral infection, although it is produced later and does have an important role in the induced response to intracellular pathogens, as we will see below. IFN-α, actually a family of several closely related proteins, and IFN-β, the product of a single gene, are synthesized by many cell types following their infection by diverse viruses. Interferon synthesis is thought to occur in response to the presence of double-stranded RNA, as synthetic double-stranded RNA is a potent inducer of interferon. Double-stranded RNA, which is not found in mammalian cells, forms the genome of some viruses and might be made as part of the infectious cycle of all viruses. Therefore, double-stranded RNA might be the common element in interferon induction.

Interferons make several contributions to defense against viral infection (Fig. 2.40). An obvious and important effect is the induction of a state of resistance to viral replication in all cells. IFN-α and IFN-β are secreted by the infected cell and then bind to a common cell-surface receptor, known as the interferon receptor, on both the infected cell and nearby cells. The interferon receptor, like many other cytokine receptors, is coupled to a Janus-family tyrosine kinase, through which it signals. This signaling pathway, which we will describe in detail in Chapter 6, rapidly induces new gene transcription as the Janus-family kinases directly phosphorylate signal-transducing activators of transcription known as STATs, which translocate to the nucleus where they activate the transcription of several different genes. In this way interferon induces the synthesis of several host cell proteins that contribute to the inhibition of viral replication. One of these is the enzyme oligoadenylate synthetase, which polymerizes ATP into a series of 2′–5′ linked oligomers (nucleotides in nucleic acids are normally linked 3′–5′). These activate an endoribonuclease that then degrades viral RNA. A second protein activated by IFN-α and IFN-β is a serine–threonine kinase called P1 kinase. This enzyme phosphorylates the eukaryotic protein synthesis initiation factor eIF-2, inhibiting translation and thus contributing to the inhibition of viral replication. Another interferon-inducible protein called Mx is known to be required for cellular resistance to influenza virus replication. Mice that lack the gene for Mx are highly susceptible to infection with the influenza virus, whereas genetically normal mice are not.

Another way interferons protect the host against viruses is by upregulating the cellular immune response to these pathogens. The adaptive immune response to viruses depends on their effective presentation to T cells as peptide fragments complexed with MHC class I molecules at the cell surface, and interferons promote this by inducing increased expression of these molecules. Interferons also activate natural killer (NK) cells to kill virus-infected cells and release cytokines. Although of lymphoid origin, NK cells do not have antigen-specific receptors and are therefore part of the innate immune system. It is not entirely clear what allows them to discriminate between infected and noninfected cells, but they possess both activating and inhibitory receptors. The latter inhibit killing when bound to normal MHC class I molecules and this means that the higher the expression of MHC class I on a cell surface, the more protected it is against destruction by NK cells. Therefore, interferons protect uninfected host cells from NK cells by upregulating class I MHC expression, while activating the NK cells to kill infected cells. Interferons also promote the release of effector cytokines by NK cells, as we will see in the next section.

2-26. Natural killer cells are activated by interferons and macrophage-derived cytokines to serve as an early defense against certain intracellular infections

Natural killer cells (NK cells) develop in the bone marrow from the common lymphoid progenitor cell and circulate in the blood. They are larger than T and B lymphocytes, have distinctive cytoplasmic granules, and are functionally identified by their ability to kill certain lymphoid tumor cell lines in vitro without the need for prior immunization or activation. The mechanism of NK cell killing is the same as that used by the cytotoxic T cells generated in an adaptive immune response; cytotoxic granules are released onto the surface of the bound target cell, and the effector proteins they contain penetrate the cell membrane and induce programmed cell death. However, NK cell killing is triggered by invariant receptors, and their known function in host defense is in the early phases of infection with several intracellular pathogens, particularly herpes viruses, the protozoan parasite Leishmania, and the bacterium Listeria monocytogenes.

NK cells are activated in response to interferons or macrophage-derived cytokines. Although NK cells that can kill sensitive targets can be isolated from uninfected individuals, this activity is increased by between twentyfold and one hundredfold when NK cells are exposed to IFN-α and IFN-β or to the NK cell-activating factor IL-12, which is one of the cytokines produced early in many infections. Activated NK cells serve to contain virus infections while the adaptive immune response generates antigen-specific cytotoxic T cells that can clear the infection (Fig. 2.41). At present the only clue to the physiological function of NK cells in humans comes from a rare patient deficient in NK cells who proved highly susceptible to early phases of herpes virus infection.

IL-12, in synergy with TNF-α, can also elicit the production of large amounts of IFN-γ by NK cells, and this secreted IFN-γ is crucial in controlling some infections before T cells have been activated to produce this cytokine. One example is the response to Listeria monocytogenes. Mice that lack T and B lymphocytes are initially quite resistant to this pathogen; however, antibody-mediated depletion of NK cells or neutralization of TNF-α or IFN-γ or their receptors renders these mice highly susceptible, so that they die a few days after infection before an adaptive immune response can be mounted.

2-27. NK cells possess receptors for self molecules that inhibit their activation against uninfected host cells

If NK cells are to mediate host defense against infection with viruses and other pathogens, they must have some mechanism for distinguishing infected from uninfected cells. Exactly how this is achieved has not yet been worked out, but recognition of ‘altered self’ is thought to be involved. NK cells have two types of surface receptor that control their cytotoxic activity. One type is an ‘activating receptor:’ it triggers killing by the NK cell. Several types of receptor provide this activation signal, including calcium-binding C-type lectins that recognize a wide variety of carbohydrate ligands present on many cells. A second set of receptors inhibit activation, and prevent NK cells from killing normal host cells. These ‘inhibitory receptors’ are specific for MHC class I alleles, which helps to explain why NK cells selectively kill target cells bearing low levels of MHC class I molecules. Altered expression of MHC class I molecules may be a common feature of cells infected by intracellular pathogens, as many of these have developed strategies to interfere with the ability of MHC class I molecules to capture and display peptides to T cells. Thus, one possible mechanism by which NK cells distinguish infected from uninfected cells is by recognizing alterations in MHC class I expression (Fig. 2.42). Another is that they recognize changes in cell-surface glycoproteins induced by viral or bacterial infection.

In mice, inhibitory receptors on NK cells are encoded by a multigene family of C-type lectins called Ly49. Different Ly49 receptors recognize different MHC class I alleles and are differentially expressed on different subsets of NK cells. Some NK cells express Ly49 receptors specific for nonself MHC alleles, but each cell expresses at least one receptor that can recognize an MHC class I allele expressed by the host. In humans, there are inhibitory receptors that recognize distinct HLA-B and HLA-C alleles (these are MHC class I alleles encoded by the B and C loci of the human MHC or Human Leukocyte Antigen gene complex). Although the MHC class I molecules of humans and mice are very similar, these human NK receptors are structurally different from those of the mouse, being members of the immunoglobulin gene superfamily; they are usually called p58 and p70, or killer inhibitory receptors (KIRs). In addition, human NK cells express a heterodimer of two C-type lectins, called CD94 and NKG2. The CD94:NKG2 receptor is also found in mice, and interacts with nonpolymorphic MHC-like molecules, HLA-E in man and Qa-1 in mice, that bind the leader peptides of other MHC class I molecules; thus this receptor may be sensitive to the presence of several different MHC class I alleles. Other inhibitory NK receptors specific for the products of the MHC class I loci are rapidly being defined, and all are members of either the immunoglobulin-like KIR family or the Ly49-like C-type lectins.

Signaling by the inhibitory NK receptors suppresses the killing activity of NK cells. This means that NK cells will not kill healthy genetically identical cells with normal expression of MHC class I molecules, such as the other cells of the body. Virus-infected cells, however, can become susceptible to killing by NK cells by a variety of mechanisms. First, some viruses inhibit all protein synthesis in their host cells, so synthesis of MHC class I proteins would be blocked in infected cells, even while being augmented by interferon in uninfected cells. The reduced level of MHC class I expression in infected cells would make them correspondingly less able to inhibit NK cells through their MHC-specific receptors, and therefore more susceptible to killing. Second, some viruses can selectively prevent the export of MHC class I molecules, which might allow the infected cell to evade recognition by the cytotoxic T cells of the adaptive immune response but would make it susceptible to killing by NK cells. There is also evidence that NK cells can detect the changes in MHC class I molecules that occur when they form complexes with peptides from proteins synthesized as a result of infection, instead of the self peptides from the proteins normally made by the cell. It is not known whether these peptides are recognized directly or whether they alter MHC conformation. Finally, virus infection alters the glycosylation of cellular proteins, perhaps allowing recognition by activating receptors to dominate or removing the normal ligand for the inhibitory receptors. Either of these last two mechanisms could allow infected cells to be detected even when the level of MHC class I expression had not been altered.

Clearly much remains to be learned about this innate mechanism of cytotoxic attack and its physiological relevance. The role of MHC molecules in allowing NK cells to detect intracellular infections is of particular interest as these same molecules govern the response of T cells to intracellular pathogens. It is possible that NK cells, which use a diverse set of nonclonotypic receptors to detect altered MHC, represent the modern remnants of the evolutionary forebears of T cells, which evolved rearranging genes that encode a vast repertoire of antigen-specific receptors geared to recognizing altered MHC.

2-28. Several lymphocyte subpopulations and ‘natural antibodies’ behave like intermediates between adaptive and innate immunity

Receptor gene rearrangements are a defining characteristic of the lymphocytes of the adaptive immune system, and allow the generation of an infinite variety of receptors, each expressed by a different individual T or B cell (see Section 1-10). However, there are several minor lymphocyte subsets that express only a very limited diversity of receptors, encoded by a few common rearrangements. These lymphocytes do not need to undergo clonal expansion before responding effectively to the antigens they recognize, and therefore behave like intermediates between adaptive and innate immunity.

One such group of cells is the intraepithelial subset of γ:δ T cells. The γ:δ T cells are themselves a minor subset of T cells that express receptors that are distinct from the α:β receptors found on the majority of T cells involved in adaptive immunity. They were discovered as a consequence of having immunoglobulin-like receptors encoded by rearranged genes and their function remains obscure. One of their most striking features is their division into two highly distinct sets of cells. One set of γ:δ T cells is found in the lymphoid tissue of all vertebrates and, like B cells and α:β T cells, they display highly diversified receptors. By contrast, intraepithelial γ:δ T cells occur variably in different vertebrates, and commonly display receptors of very limited diversity, particularly in the skin and the female reproductive tract of mice, where the γ:δ T cells are essentially homogeneous in any one site. On the basis of this limited diversity of epithelial γ:δ T-cell receptors and their limited recirculatory behavior, it has been proposed that intraepithelial γ:δ T cells may recognize ligands that are derived from the epithelium in which they reside, but which are expressed only when a cell has become infected. Candidate ligands are heat-shock proteins, MHC class IB molecules, and unorthodox nucleotides and phospholipids, for all of which there is evidence of recognition by γ:δ T cells. Unlike α:β T cells, γ:δ T cells do not generally recognize antigen as peptides presented by MHC molecules; instead they seem to recognize their target antigens directly, and could potentially recognize and respond rapidly to molecules expressed by many different cell types. Recognition of molecules expressed as a consequence of infection, rather than of pathogen-specific antigens themselves, would distinguish γ:δ T cells from other lymphocytes and arguably place them at the intersection between innate and adaptive immunity. However, several recent studies of mice deficient in γ:δ T cells have revealed exaggerated responses to various pathogens and even to self tissues, rather than deficiencies in pathogen control and rejection. This has led to the suggestion that at least some γ:δ T cells have a regulatory role in modulating immune responses, a function that would be consistent with their demonstrated ability to secrete regulatory cytokines when activated. Which aspects of the phenotype of γ:δ-deficient mice are attributable to which subset of γ:δ T cells remains to be clarified.

Another subset of lymphocytes that express a limited diversity of receptors is the B-1 subset of B cells. B cells of this lineage are distinguished by the cell-surface protein CD5 and have properties quite distinct from those of conventional B cells that mediate adaptive humoral immunity. These so-called CD5 B cells, or B-1 cells, are in many ways analogous to epithelial γ:δ T cells: they arise early in ontogeny, they use a distinctive and limited set of gene rearrangements to make their receptors, they are self-renewing in the periphery, and they are the predominant lymphocyte in a distinctive microenvironment, the peritoneal cavity.

B-1 cells seem to make antibody responses mainly to polysaccharide antigens and can produce antibodies of the IgM class without needing T-cell help (Fig. 2.43). Although these responses can be augmented by T cells, they appear within 48 hours of the exposure to antigen, and the T cells involved are therefore not part of an antigen-specific adaptive immune response. The lack of an antigen-specific interaction with helper T cells might explain why immunological memory is not generated: repeated exposures to the same antigen elicit similar or decreased responses with each exposure. Thus, these responses, although generated by lymphocytes with rearranging receptors, resemble innate rather than adaptive immune responses.

As with γ:δ T cells, the precise role of B-1 cells in host defense is uncertain. Mice that are deficient in B-1 cells are more susceptible to infection with Streptococcus pneumoniae because they fail to produce an antibody against the phospholipid headgroup phosphorylcholine that effectively protects against this organism. A significant fraction of the B-1 cells can make antibodies of this specificity, and because no antigen-specific T-cell help is required, a potent response can be produced early in infection with this pathogen. Whether human B-1 cells have the same role is uncertain.

In terms of evolution, it is interesting to note that γ:δ T cells seem to defend the body surfaces, whereas B-1 cells defend the body cavity. Both cell types are relatively limited in their range of specificities and in the efficiency of their responses. It is possible that these two cell types represent a transitional phase in the evolution of the adaptive immune response, guarding the two main compartments of primitive organisms—the epithelial surfaces and the body cavity. It is not yet clear whether they are still critical to host defense or whether they represent an evolutionary relic. Nevertheless, as each cell type is prominent in certain sites in the body and contributes to certain responses, they must be incorporated into our thinking about host defense.

Finally, there is a collection of antibodies known as ‘natural antibody.’ This ‘natural IgM’ is encoded by rearranged antibody genes that have not undergone somatic mutation. It makes up a considerable amount of the IgM circulating in humans and does not appear to be a result of an antigen-specific adaptive immune response to infection. It has a low affinity for many microbial pathogens, and is very highly cross-reactive, even binding to some self molecules. It is unknown whether this natural antibody has any role in host defense or which type of B cells produce it. Furthermore, it is not known whether it is produced in response to the normal flora of the epithelial surfaces of the body or in response to self. However, it might play a role in host defense by binding to the earliest infecting pathogens and clearing them before they become dangerous.

Summary

Innate immunity can use a variety of induced effector mechanisms to clear an infection or, failing that, to hold it in check until the pathogen can be recognized by the adaptive immune system. These effector mechanisms are all regulated by germline-encoded receptor systems that are able to discriminate between noninfected self and infectious nonself ligands. Thus the phagocytes’ ability to discriminate between self and pathogen controls its release of pro-inflammatory chemokines and cytokines that act together to recruit more phagocytic cells, especially neutrophils, which can also recognize pathogens, to the site of infection. Furthermore, cytokines released by tissue phagocytic cells induce fever, the production of acute-phase response proteins including the pathogen-binding mannan-binding lectin and the C-reactive proteins, and the mobilization of antigen-presenting cells that induce the adaptive immune response. Viral pathogens are recognized by the cells in which they replicate, leading to the production of interferons that serve to inhibit viral replication and to activate NK cells, which in turn can distinguish infected from noninfected cells. As we will see later in this book, cytokines, chemokines, phagocytic cells, and NK cells are all effector mechanisms that also are employed in an adaptive immune response that uses clonotypic receptors to target specific pathogen antigens.