9-12. Antibodies of different isotype operate in distinct places and have distinct effector functions

Pathogens most commonly enter the body across the epithelial barriers of the mucosa lining the respiratory, digestive, and urogenital tracts, or through damaged skin, and can then establish infections in the tissues. Less often, insects, wounds, or hypodermic needles introduce microorganisms directly into the blood. The body's mucosal surfaces, tissues, and blood are all protected by antibodies from such infections; these antibodies serve to neutralize the pathogen or promote its elimination before it can establish a significant infection. Antibodies of different isotypes are adapted to function in different compartments of the body. Because a given V region can become associated with any C region through isotype switching (see Section 4-16), the progeny of a single B cell can produce antibodies, all specific for the same eliciting antigen, that provide all of the protective functions appropriate for each body compartment.

The first antibodies to be produced in a humoral immune response are always IgM, because IgM can be expressed without isotype switching (see Figs 4.20 and 9.8). These early IgM antibodies are produced before B cells have undergone somatic hypermutation and therefore tend to be of low affinity. IgM molecules, however, form pentamers whose 10 antigen-binding sites can bind simultaneously to multivalent antigens such as bacterial capsular polysaccharides. This compensates for the relatively low affinity of the IgM monomers by multipoint binding that confers high overall avidity. As a result of the large size of the pentamers, IgM is mainly found in the blood and, to a lesser extent, the lymph. The pentameric structure of IgM makes it especially effective in activating the complement system, as we will see in the last part of this chapter. Infection of the bloodstream has serious consequences unless it is controlled quickly, and the rapid production of IgM and its efficient activation of the complement system are important in controlling such infections. Some IgM is also produced in secondary and subsequent responses, and after somatic hypermutation, although other isotypes dominate the later phases of the antibody response.

Antibodies of the other isotypes—IgG, IgA, and IgE—are smaller in size and diffuse easily out of the blood into the tissues. Although IgA can form dimers, as we saw in Chapter 4, IgG and IgE are always monomeric. The affinity of the individual antigen-binding sites for their antigen is therefore critical for the effectiveness of these antibodies, and most of the B cells expressing these isotypes have been selected for increased affinity of antigen-binding in germinal centers. IgG is the principal isotype in the blood and extracellular fluid, whereas IgA is the principal isotype in secretions, the most important being those of the mucus epithelium of the intestinal and respiratory tracts. Whereas IgG efficiently opsonizes pathogens for engulfment by phagocytes and activates the complement system, IgA is a less potent opsonin and a weak activator of complement. This distinction is not surprising, as IgG operates mainly in the body tissues, where accessory cells and molecules are available, whereas IgA operates mainly on epithelial surfaces where complement and phagocytes are not normally present, and therefore functions chiefly as a neutralizing antibody.

Finally, IgE antibody is present only at very low levels in blood or extracellular fluid, but is bound avidly by receptors on mast cells that are found just beneath the skin and mucosa, and along blood vessels in connective tissue. Antigen binding to this IgE triggers mast cells to release powerful chemical mediators that induce reactions, such as coughing, sneezing, and vomiting, that can expel infectious agents, as will be discussed below when we describe the receptors that bind immunoglobulin C regions and engage effector functions. The distribution and main functions of antibodies of the different isotypes are summarized in Fig. 9.19.

Figure 9.19

Each human immuno-globulin isotype has specialized functions and a unique distribution. The major effector functions of each isotype (+ + +) are shaded in dark red, whereas lesser functions (+ +) are shown in dark pink, and very minor functions (+) in (more...)

9-13. Transport proteins that bind to the Fc regions of antibodies carry particular isotypes across epithelial barriers

IgA-secreting plasma cells are found predominantly in the connective tissue called the lamina propria, which lies immediately below the basement membrane of many surface epithelia. From there, the IgA antibodies can be transported across the epithelium to its external surface, for example, to the lumen of the gut or the bronchi. IgA antibody synthesized in the lamina propria is secreted as a dimeric IgA molecule associated with a single J chain (see Fig. 4.23). This polymeric form of IgA binds specifically to the poly-Ig receptor, which is present on the basolateral surfaces of the overlying epithelial cells (Fig. 9.20). When the poly-Ig receptor has bound a molecule of dimeric IgA, the complex is internalized and carried through the cytoplasm of the epithelial cell in a transport vesicle to its luminal surface. This process is called transcytosis. At the apical or luminal surface of the epithelial cell, the poly-Ig receptor is cleaved enzymatically, releasing the extracellular portion of the receptor still attached to the Fc region of the dimeric IgA. This fragment of receptor, called the secretory component, may help to protect the IgA dimer from proteolytic cleavage. Some molecules of dimeric IgA diffuse from the lamina propria into the extracellular spaces of the tissues, draining into the bloodstream before being excreted into the gut via the bile. Therefore, it is not surprising that patients with obstructive jaundice, a condition in which bile is not excreted, show a marked increase in dimeric IgA in the plasma.

Figure 9.20

Transcytosis of IgA antibody across epithelia is mediated by the poly-Ig receptor, a specialized transport protein. Most IgA antibody is synthesized in plasma cells lying just beneath epithelial basement membranes of the gut, the respiratory epithelia, the (more...)

The principal sites of IgA synthesis and secretion are the gut, the respiratory epithelium, the lactating breast, and various other exocrine glands such as the salivary and tear glands. It is believed that the primary functional role of IgA antibodies is to protect epithelial surfaces from infectious agents, just as IgG antibodies protect the extracellular spaces of the internal tissues. IgA antibodies prevent the attachment of bacteria or toxins to epithelial cells and the absorption of foreign substances, and provide the first line of defense against a wide variety of pathogens. Newborn infants are especially vulnerable to infection, having had no prior exposure to the microbes in the environment they enter at birth. IgA antibodies are secreted in breast milk and are thereby transferred to the gut of the newborn infant, where they provide protection from newly encountered bacteria until the infant can synthesize its own protective antibody.

IgA is not the only protective antibody a mother passes on to her baby. Maternal IgG is transported across the placenta directly into the bloodstream of the fetus during intrauterine life; human babies at birth have as high a level of plasma IgG as their mothers, and with the same range of antigen specificities. The selective transport of IgG from mother to fetus is due to an IgG transport protein in the placenta, FcRn, which is closely related in structure to MHC class I molecules. Despite this similarity, FcRn binds IgG quite differently from the binding of peptide to MHC class I, as its peptide-binding groove is occluded. It binds to the Fc portion of IgG molecules (Fig. 9.21). Two molecules of FcRn bind one molecule of IgG, bearing it across the placenta. In some rodents, FcRn also delivers IgG to the circulation of the neonate from the gut lumen. Maternal IgG is ingested by the newborn animal in its mother's milk and colostrum, the protein-rich fluid secreted by the early postnatal mammary gland. In this case, FcRn transports the IgG from the lumen of the neonate gut into the blood and tissues. Interestingly, FcRn is also found in adults in the gut and liver and on endothelial cells. Its function in adults is to regulate the levels of IgG in serum and other body fluids, which it does by binding circulating antibody, endocytosing it, and then recycling to the cell surface.

Figure 9.21

FcRn binds to the Fc portion of IgG. The structure of a molecule of FcRn (white) bound to one chain of the Fc portion of IgG (blue) is shown. FcRn transports IgG molecules across the placenta in humans and also across the gut in rats and mice. It also (more...)

By means of these specialized transport systems, mammals are supplied from birth with antibodies against pathogens common in their environments. As they mature and make their own antibodies of all isotypes, these are distributed selectively to different sites in the body (Fig. 9.22). Thus, throughout life, isotype switching and the distribution of isotypes through the body provide effective protection against infection in extracellular spaces.

Figure 9.22

Immunoglobulin isotypes are selectively distributed in the body. IgG and IgM predominate in plasma, whereas IgG and monomeric IgA are the major isotypes in extracellular fluid within the body. Dimeric IgA predominates in secretions across epithelia, including (more...)

9-14. High-affinity IgG and IgA antibodies can neutralize bacterial toxins

Many bacteria cause disease by secreting proteins called toxins, which damage or disrupt the function of the host's cells (Fig. 9.23). To have an effect, a toxin must interact specifically with a molecule that serves as a receptor on the surface of the target cell. In many toxins, the receptor-binding domain is on one polypeptide chain whereas the toxic function is carried by a second chain. Antibodies that bind to the receptor-binding site on the toxin molecule can prevent the toxin from binding to the cell and thus protect the cell from attack (Fig. 9.24). Antibodies that act in this way to neutralize toxins are referred to as neutralizing antibodies.

Figure 9.23

Many common diseases are caused by bacterial toxins. These toxins are all exotoxins—proteins secreted by the bacteria. High-affinity IgG and IgA antibodies protect against these toxins. Bacteria also have nonsecreted endotoxins, such as lipopolysaccharide, (more...)

Figure 9.24

Neutralization of toxins by IgG antibodies protects cells from their damaging action. Many bacteria (as well as venomous insects and snakes) cause their damaging effects by elaborating toxic proteins (see Fig. 9.23). These toxins are usually composed (more...)

Most toxins are active at nanomolar concentrations: a single molecule of diphtheria toxin can kill a cell. To neutralize toxins, therefore, antibodies must be able to diffuse into the tissues and bind the toxin rapidly and with high affinity. The ability of IgG antibodies to diffuse easily throughout the extracellular fluid and their high affinity make these the principal neutralizing antibodies for toxins found in tissues. IgA antibodies similarly neutralize toxins at the mucosal surfaces of the body.

Diphtheria and tetanus toxins are two bacterial toxins in which the toxic and receptor-binding functions are on separate protein chains. It is therefore possible to immunize individuals, usually as infants, with modified toxin molecules in which the toxic chain has been denatured. These modified toxins, called toxoids, lack toxic activity but retain the receptor-binding site. Thus, immunization with the toxoid induces neutralizing antibodies that protect against the native toxin.

With some insect or animal venoms that are so toxic that a single exposure can cause severe tissue damage or death, the adaptive immune response is too slow to be protective. Exposure to these venoms is a rare event and protective vaccines have not been developed for use in humans. Instead, neutralizing antibodies are generated by immunizing other species, such as horses, with insect and snake venoms to produce anti-venom antibodies (antivenins) for use in protecting humans. Transfer of antibodies in this way is known as passive immunization (see Appendix I, Section A-37).

9-15. High-affinity IgG and IgA antibodies can inhibit the infectivity of viruses

Animal viruses infect cells by binding to a particular cell-surface receptor, often a cell-type-specific protein that determines which cells they can infect. The hemagglutinin of influenza virus, for example, binds to terminal sialic acid residues on the carbohydrates of glycoproteins present on epithelial cells of the respiratory tract. It is known as hemagglutinin because it recognizes and binds to similar sialic acid residues on chicken red blood cells and agglutinates these red blood cells. Antibodies to the hemagglutinin can prevent infection by the influenza virus. Such antibodies are called virus-neutralizing antibodies and, as with the neutralization of toxins, high-affinity IgA and IgG antibodies are particularly important.

Many antibodies that neutralize viruses do so by directly blocking viral binding to surface receptors (Fig. 9.25). However, viruses are sometimes successfully neutralized when only a single molecule of antibody is bound to a virus particle that has many receptor-binding proteins on its surface. In these cases, the antibody must cause some change in the virus that disrupts its structure and either prevents it from interacting with its receptors or interferes with the fusion of the virus membrane with the cell surface after the virus has engaged its surface receptor.

Figure 9.25

Viral infection of cells can be blocked by neutralizing antibodies. For a virus to multiply within a cell, it must introduce its genes into the cell. The first step in entry is usually the binding of the virus to a receptor on the cell surface. For enveloped (more...)

9-16. Antibodies can block the adherence of bacteria to host cells

Many bacteria have cell-surface molecules called adhesins that enable them to bind to the surface of host cells. This adherence is critical to the ability of these bacteria to cause disease, whether they subsequently enter the cell, as do some pathogens such as Salmonella species, or remain attached to the cell surface as extracellular pathogens (Fig. 9.26). Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has a cellsurface protein known as pilin. Pilin enables the bacterium to adhere to the epithelial cells of the urinary and reproductive tracts and is essential to its infectivity. Antibodies against pilin can inhibit this adhesive reaction and prevent infection.

Figure 9.26

Antibodies can prevent attachment of bacteria to cell surfaces. Many bacterial infections require an interaction between the bacterium and a cell-surface receptor. This is particularly true for infections of mucosal surfaces. The attachment process involves (more...)

IgA antibodies secreted onto the mucosal surfaces of the intestinal, respiratory, and reproductive tracts are particularly important in preventing infection by preventing the adhesion of bacteria, viruses, or other pathogens to the epithelial cells lining these surfaces. The adhesion of bacteria to cells within tissues can also contribute to pathogenesis, and IgG antibodies against adhesins can protect from damage much as IgA antibodies protect at mucosal surfaces.

9-17. Antibody:antigen complexes activate the classical pathway of complement by binding to C1q

Another way in which antibodies can protect against infection is by activation of the cascade of complement proteins. We have described these proteins in Chapter 2, as they can also be activated on pathogen surfaces in the absence of antibody, as part of the innate immune response. Complement activation proceeds via a series of proteolytic cleavage reactions, in which inactive components, present in plasma, are cleaved to form proteolytic enzymes that attach covalently to the pathogen surface. All known pathways of complement activation converge to generate the same set of effector actions: the pathogen surface or immune complex is coated with covalently attached fragments (principally C3b) that act as opsonins to promote uptake and removal by phagocytes. At the same time, small peptides with inflammatory and chemotactic activity are released (principally C5a) so that phagocytes are recruited to the site. In addition, the terminal complement components can form a membrane-attack complex that damages some bacteria.

Antibodies initiate complement activation by a pathway known as the classical pathway because it was the first pathway of complement activation to be discovered. The full details of this pathway, and of the other two known pathways of complement activation, are given in Chapter 2, but we will describe here how antibody is able to initiate the classical pathway after binding to pathogen, or after forming immune complexes.

The first component of the classical pathway of complement activation is C1, which is a complex of three proteins called C1q, C1r, and C1s. Two molecules each of C1r and C1s are bound to each molecule of C1q (see Fig. 2.10). Complement activation is initiated when antibodies attached to the surface of a pathogen bind C1q. C1q can be bound by either IgM or IgG antibodies but, because of the structural requirements of binding to C1q, neither of these antibody isotypes can activate complement in solution; the cascade is initiated only when the antibodies are bound to multiple sites on a cell surface, normally that of a pathogen.

The C1q molecule has six globular heads joined to a common stem by long, filamentous domains that resemble collagen molecules; the whole C1q complex has been likened to a bunch of six tulips held together by the stems. Each globular head can bind to one Fc domain, and binding of two or more globular heads activates the C1q molecule. In plasma, the pentameric IgM molecule has a planar conformation that does not bind C1q (Fig. 9.27, left panel); however, binding to the surface of a pathogen deforms the IgM pentamer so that it looks like a staple (see Fig. 9.27, right panel), and this distortion exposes binding sites for the C1q heads. Although C1q binds with low affinity to some subclasses of IgG in solution, the binding energy required for C1q activation is achieved only when a single molecule of C1q can bind two or more IgG molecules that are held within 30–40 nm of each other as a result of binding antigen. This requires many molecules of IgG to be bound to a single pathogen. For this reason, IgM is much more efficient in activating complement than is IgG. The binding of C1q to a single bound IgM molecule, or to two or more bound IgG molecules, leads to the activation of an enzymatic activity in C1r, triggering the complement cascade as shown schematically in Fig. 9.28. This translates antibody binding into the activation of the complement cascade, which, as we learned in Chapter 2, can also be triggered by direct binding of C1q to the pathogen surface.

Figure 9.27

The two conformations of IgM. The left panel shows the planar conformation of soluble IgM; the right panel shows the staple conformation of IgM bound to a bacterial flagellum. Photographs (× 760,000) courtesy of K.H. Roux.

Figure 9.28

The classical pathway of complement activation is initiated by binding of C1q to antibody on a surface such as a bacterial surface. In the left panels, one molecule of IgM, bent into the ‘staple’ conformation by binding several identical (more...)

9-18. Complement receptors are important in the removal of immune complexes from the circulation

Many small soluble antigens form antibody:antigen complexes known as immune complexes that contain too few molecules of IgG to be readily bound to the Fcγ receptors we will discuss in the next part of the chapter. These antigens include toxins bound by neutralizing antibodies and debris from dead microorganisms. Such immune complexes are found after most infections and are removed from the circulation through the action of complement. The soluble immune complexes trigger their own removal by activating complement, again through the binding of C1q, leading to the covalent binding of the activated components C4b and C3b to the complex, which is then cleared from the circulation by the binding of C4b and C3b to CR1 on the surface of erythrocytes. The erythrocytes transport the bound complexes of antigen, antibody, and complement to the liver and spleen. Here, macrophages bearing CR1 and Fc receptors remove the complexes from the erythrocyte surface without destroying the cell, and then degrade them (Fig. 9.29). Even larger aggregates of particulate antigen and antibody can be made soluble by activation of the classical complement pathway, and then removed by binding to complement receptors.

Figure 9.29

Erythrocyte CR1 helps to clear immune complexes from the circulation. CR1 on the erythrocyte surface has an important role in the clearance of immune complexes from the circulation. Immune complexes bind to CR1 on erythrocytes, which transport them to (more...)

Immune complexes that are not removed tend to deposit in the basement membranes of small blood vessels, most notably those of the renal glomerulus where the blood is filtered to form urine. Immune complexes that pass through the basement membrane of the glomerulus bind to the complement receptor CR1 on the renal podocytes, cells that lie beneath the basement membrane. The functional significance of these receptors in the kidney is unknown; however, they play an important part in the pathology of some autoimmune diseases.

In the autoimmune disease systemic lupus erythematosus (Systemic Lupus Erythematosus, in Case Studies in Immunology, see Preface for details), which we will describe in Chapter 13, excessive levels of circulating immune complexes cause huge deposits of antigen, antibody, and complement on the podocytes, damaging the glomerulus; kidney failure is the principal danger in this disease. Immune complexes can also be a cause of pathology in patients with deficiencies in the early components of complement. Such patients do not clear immune complexes effectively and they also suffer tissue damage, especially in the kidneys, in a similar way.

Summary

The T-cell dependent antibody response begins with IgM secretion but quickly progresses to the production of all the different isotypes. Each isotype is specialized both in its localization in the body and in the functions it can perform. IgM antibodies are found mainly in blood; they are pentameric in structure. IgM is specialized to activate complement efficiently upon binding antigen. IgG antibodies are usually of higher affinity and are found in blood and in extracellular fluid, where they can neutralize toxins, viruses, and bacteria, opsonize them for phagocytosis, and activate the complement system. IgA antibodies are synthesized as monomers, which enter blood and extracellular fluids, or as dimeric molecules in the lamina propria of various epithelia. IgA dimers are selectively transported across these epithelia into sites such as the lumen of the gut, where they neutralize toxins and viruses and block the entry of bacteria across the intestinal epithelium. Most IgE antibody is bound to the surface of mast cells that reside mainly just below body surfaces; antigen binding to this IgE triggers local defense reactions. Thus, each of these isotypes occupies a particular site in the body and has a particular role in defending the body against extracellular pathogens and their toxic products. Antibodies can accomplish this by direct interactions with pathogens or their products, for example by binding to active sites of toxins and neutralizing them or by blocking their ability to bind to host cells through specific receptors. When antibodies of the appropriate isotype bind to antigens, they can activate the classical pathway of complement, which leads to the elimination of the pathogen by the various mechanisms described in Chapter 2. Soluble immune complexes of antigen and antibody also fix complement and are cleared from the circulation via complement receptors on red blood cells.