The three major components of the body’s immune system are: B lymphocytes (B cells), T lymphocytes (T cells), and the innate immune system. B lymphocytes are mainly derived from bone marrow cells, while T lymphocytes originate from the thymus. The B cells are responsible for the synthesis of circulating, humoral antibodies, also known as immunoglobulins. The T cells are involved in a variety of important cell-mediated immunologic processes such as graft rejection, hypersensitivity reactions, and defense against malignant cells and many viruses. B and T cells respond in an adaptive manner, developing a targeted response for each invader encountered. The innate immune system defends against infection in a nonspecific manner. It contains a variety of cells such as phagocytes, neutrophils, natural killer cells, and others that will be discussed in Chapter 54.
Immunoglobulins Are Comprised of Multiple Polypeptide Chains
Immunoglobulins (Ig) are oligomeric proteins whose individual subunits traditionally have been classified as heavy (H) or light (L) based on their migration during SDS-polyacrylamide gel electrophoresis. Human immunoglobulins can be grouped into five classes abbreviated as IgA, IgD, IgE, IgG, and IgM (Table 52–8). The biologic functions of each class are summarized in Table 52–9. The most abundant of the five, IgG, consist of two identical light chains (23 kDa) and two identical heavy chains (53–75 kDa) linked together by a network of disulfide bonds. The L chains and H chains are synthesized as separate polypeptides that are subsequently assembled within the B cell or plasma cell into mature immunoglobulin molecules, all of which are glycoproteins.
TABLE 52–8Properties of Human Immunoglobulins ||Download (.pdf) TABLE 52–8 Properties of Human Immunoglobulins
|Property ||IgG ||IgA ||IgM ||IgD ||IgE |
|Percentage of total immunoglobulin in serum (approximate) ||75 ||15 ||9 ||0.2 ||0.004 |
|Serum concentration (mg/dL) (approximate) ||1000 ||200 ||120 ||3 ||0.05 |
|Sedimentation coefficient ||7S ||7S or 11Sa ||19S ||7S ||8S |
|Molecular weight (×1000) ||150 ||170 or 400a ||900 ||180 ||190 |
|Structure ||Monomer ||Monomer or dimer ||Monomer or pentamer ||Monomer ||Monomer |
|H-chain symbol ||γ ||α ||μ ||δ ||ε |
|Complement fixation ||+ ||— ||+ ||— ||— |
|Transplacental passage ||+ ||— ||— ||? ||— |
|Mediation of allergic responses ||— ||— ||— ||— ||+ |
|Found in secretions ||— ||+ ||— ||— ||— |
|Opsonization ||+ ||— ||—b ||— ||— |
|Antigen receptor on B cell ||— ||— ||+ ||? ||— |
|Polymeric form contains J chain ||— ||+ ||+ ||— ||— |
TABLE 52–9Major Functions of Immunoglobulins ||Download (.pdf) TABLE 52–9 Major Functions of Immunoglobulins
|Immunoglobulin ||Major Functions |
|IgG ||Main antibody in the secondary response. Opsonizes bacteria, making them easier to phagocytose. Fixes complement, which enhances bacterial killing. Neutralizes bacterial toxins and viruses. Crosses the placenta. |
|IgA ||Secretory IgA prevents attachment of bacteria and viruses to mucous membranes. Does not fix complement. |
|IgM ||Produced in the primary response to an antigen. Fixes complement. Does not cross the placenta. Antigen receptor on the surface of B cells. |
|IgD ||Found on the surfaces of B cells where it acts as a receptor for antigen. |
|IgE ||Mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to antigen (allergen). Defends against worm infections by causing release of enzymes from eosinophils. Does not fix complement. Main host defense against helminthic infections. |
The Y-shaped configuration of the immunoglobulin core unit is illustrated by the IgG heterotetramer (L2H2) shown in Figure 52–11. Some immunoglobulins such as immune IgG exist only in the basic tetrameric structure. Others such as IgA and IgM can form higher oligomers comprised of two, three (IgA), or five (IgM) copies of the core tetrameric unit (Figure 52–12). The type of H chain determines the class of immunoglobulin and thus its effector function (see below): α (IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM). The γ chains of IgG are organized into four conserved domains: an amino terminal variable region (VH) and three constant regions (CH1, CH2, CH3). The five types of H chains are distinguished by differences in their CH regions. The μ and ε chains each have four CH domains rather than the usual three.
Structure of IgG. The molecule consists of two light (L) chains and two heavy (H) chains. Each light chain consists of a variable (VL) and a constant (CL) region. Each heavy chain consists of a variable region (VH) and a constant region that is divided into three domains (CH1, CH2, and CH3). The CH2 domain contains the complement-binding site and the CH3 domain contains a site that attaches to receptors on neutrophils and macrophages. The antigen-binding site is formed by the hypervariable regions of both the light and heavy chains, which are located in the variable regions of these chains (see Figure 50–10). The light and heavy chains are linked by disulfide bonds, and the heavy chains are also linked to each other by disulfide bonds. (Reproduced, with permission, from Parslow TG, et al (editors): Medical Immunology, 10th ed. McGraw-Hill, 2001.)
Schematic representation of serum IgA, secretory IgA, and IgM. Both IgA and IgM have a J chain, but only secretory IgA has a secretory component. Polypeptide chains are represented by thick lines; disulfide bonds linking different polypeptide chains are represented by thin lines. (Reproduced, with permission, from Parslow TG, et al (editors): Medical Immunology, 10th ed. McGraw-Hill, 2001.)
The IgG light chain can be divided into a C-terminal constant region (CL) and amino terminal variable region (VL). There are two general types of light chains, kappa (κ) and lambda (λ), which can be distinguished on the basis of structural differences in their CL regions. A given immunoglobulin molecule always contains two κ or two λ light chains—never a mixture of κ and λ. In humans, the κ chains are more common than λ chains in immunoglobulin molecules.
IgG molecules are divalent. The tip of each Y contains an antigen-binding site made up of VH and VL domains arranged together to form two sheets of antiparallel amino acids. The site on the antigen to which an antibody binds is termed an antigenic determinant, or epitope. The region between the CH1 and CH2 domains, which can be readily cleaved using the pepsin or papain (Figure 52–11), is referred to as the “hinge region.” The hinge region confers flexibility and allows both Fab arms to move independently. This facilitates binding to antigenic sites that may be variable distances apart or that are present on two different bacteria or viruses. In this way, antibody-antigen clusters can be formed whose size renders them more easily recognized and disposed of by phagocytic leukocytes. This phenomenon is commonly demonstrated in the laboratory by the formation of erythrocyte rosettes.
Variable Regions Confer Binding Specificity
The variable regions of the immunoglobulin light and heavy chains form the antigen-binding sites that dictate the amazing specificity of antibodies. As their name implies, they are quite heterogeneous. In fact, no two variable regions from different humans share identical amino acid sequences. The variable regions of the L and H chains consist of a handful of short (5-10 residue) islands called hypervariable regions interspersed within a polypeptide “sea” by relatively invariable framework regions (Figure 52–13). Hypervariable regions are also termed complementarity-determining regions (CDRs). An antigen-binding site is formed when the hypervariable regions of the H and L chains align together in three-dimensional space (tertiary structure) as projecting loops from the antibody surface.
Schematic model of an IgG molecule showing approximate positions of the hypervariable regions in heavy and light chains. The antigen-binding site is formed by these hypervariable regions. The hypervariable regions are also called complementarity-determining regions (CDRs). (Modified and reproduced, with permission, from Parslow TG, et al (editors): Medical Immunology, 10th ed. McGraw-Hill, 2001.)
Various combinations of H and L chain CDRs can give rise to multiple antibodies possessing different specificities, a feature termed combinatorial diversity. Large antigens interact with all of the CDRs of an antibody, whereas small ligands may interact with only one or a few CDRs that form a pocket or groove in the antibody molecule. The essence of antigen-antibody interactions is mutual complementarity between the surfaces of CDRs and epitopes that involve multiple noncovalent interactions such as hydrogen bonding, salt bridges, hydrophobic interactions, and van der Waal’s forces (see Chapter 2).
The Constant Regions Determine Class-Specific Effector Functions
The constant regions of the immunoglobulin molecules, particularly the CH2 and CH3 (and CH4 of IgM and IgE) located within the Fc fragment, are responsible for the class-specific effector functions of the different immunoglobulin molecules (Table 52–9, bottom part), such as complement fixation or transplacental passage.
Antibody Diversity Depends on Gene Rearrangements
The human genome contains less than 150 immunoglobulin genes. Nevertheless, each person is capable of synthesizing perhaps 1 million different antibodies, each specific for a unique antigen. Clearly, immunoglobulin expression does not follow the “one gene, one protein” paradigm. Instead, immunoglobulin diversity is generated by combinatorial mechanisms based upon mixing and rearranging a finite pool of genetic information in multiple ways (see Chapters 35 and 38).
The first source of antibody diversity is the division of the coding sequence for each immunoglobulin chain among multiple genes. Each light chain is the product of at least three separate structural genes that code for the variable region (VL), joining region (J) (bearing no relationship to the J chain of IgA or IgM), and constant region (CL), respectively. Similarly, each heavy chain is the product of at least four different genes that code for a variable region (VH) gene, a diversity region (D), a joining region (J), and a constant region (CH) gene. Each gene is present in the human genome in several versions offering the potential for the assembly of a multiplicity of combinations.
Diversity is further augmented through the action of the activation-induced cytidine deaminase (AID). By catalyzing the conversion of cytidine to uracil, AID massively increases the frequency of mutation of immunoglobulin V genes. These mutations are somatic in nature, ie, unique to a differentiated cell rather than to a germline cell. Consequently, each activation of AID generates new subpopulations of B cells that harbor unique mutations of their V genes, causing each to synthesize immunoglobulins of differing antigen specificity. In some pathologic states, the mutagenic action of AID can lead to the generation of autoantibodies that target the body’s endogenous components, a phenomenon termed autoimmunity.
A third mechanism for generating antibodies targeting novel antigens is junctional diversity. This refers to the addition or deletion of random numbers of nucleotides that takes place when certain gene segments are joined together. As is the case with AID, the mutations generated by junctional diversity are somatic in nature.
Class (Isotype) Switching Occurs During Immune Responses
In most humoral immune responses, antibodies of different classes are generated that possess identical antigen specificities. Each class appears in a specific chronologic order in response to the immunogen (immunizing antigen). For instance, antibodies of the IgM class normally precede molecules of the IgG class. The transition from the synthesis of one class to another is designated class or isotype switching. Switching involves the combining of a given immunoglobulin light chain with different heavy chains. Whereas a newly synthesized light chain will initially be mated with a μ chain to generate a specific IgM molecule, over time the same antigen-specific light chain will be mated with a γ chain. This γ chain will, however, possess an identical VH region to that of the μ chain, thus generating an IgG whose antigen specificity is identical to that of the original IgM molecule. The same light chain can also combine with an α heavy chain, again containing the identical VH region, to form an IgA molecule with identical antigen specificity. Immunoglobulin molecules of different classes that possess identical variable domains and antigen specificity are said to share an idiotype. (Idiotypes are the antigenic determinants formed by the specific amino acids in the hypervariable regions.)
Monoclonal Antibodies Are an Important Research Tool
Antibodies have emerged as a major tool in biomedical research, diagnosis, and treatment. Originally, the production of antibodies against a selected antigen required that the antigen be injected into a host animal, such as a rabbit or goat, and serum containing plasma immunoglobulins that included (hopefully) antibodies against the antigen of interest obtained. When an antigen is injected into an animal, the resulting antibodies are produced by a mixture of B cells that synthesize antibodies directed against different sites (epitopes or determinants) on the antigen. Antibodies produced in animal hosts are thus heterogeneous or polyclonal in nature. Moreover, unless subjected to costly affinity purification, serum immunoglobulins also contain antibodies against many thousands of antigens to which the host animal has been exposed during its lifetime.
Homogenous monoclonal antibodies targeting a single epitope, and that are free from other contaminating immunoglobulins, can be generated in the laboratory. Typically, B cells are obtained from the spleen of a mouse (or other suitable animal) previously injected with an antigen or mixture of antigens (eg, foreign cells). The B cells are mixed with mouse myeloma cells and exposed to polyethylene glycol, which causes cell fusion. The product of this fusion is a permanent cell line called a hybridoma capable of providing a continuous supply of monoclonal antibodies. Figure 52–14 summarizes the principles involved in generating hybridoma cells. By plating highly diluted cell mixtures on a selective, hypoxanthine-aminopterin-thymidine (HAT)-containing medium, homogenous, clonal hybridoma lines originating from a single cell can be isolated. By identifying lines that secrete a monoclonal antibody specific for the antigen of choice, it is possible to obtain a battery of monoclonal antibodies specific for individual components of the immunogenic mixture or different epitopes on a single antigen. The hybridoma cells can be frozen and stored and subsequently thawed when more of the antibody is required; this ensures its long-term supply.
Scheme of production of a hybridoma cell. The myeloma cells are immortalized, do not produce antibody, and are HGPRT− (rendering the salvage pathway of purine synthesis [see Chapter 33] inactive). The B cells are not immortalized, each produces a specific antibody, and they are HGPRT+. Polyethylene glycol (PEG) stimulates cell fusion. The resulting hybridoma cells are immortalized (via the parental myeloma cells), produce antibody, and are HGPRT+ (both latter properties gained from the parental B cells). Any remaining B cells will die because they are not immortalized. In the presence of HAT, the myeloma cells will also die since the aminopterin in HAT suppresses purine synthesis via the de novo pathway by inhibiting reutilization of tetrahydrofolate (see Chapter 33). However, the hybridoma cells will survive, grow (because they are HGPRT+), and—if cloned—produce monoclonal antibody. (HAT, hypoxanthine, aminopterin, and thymidine; HGPRT, hypoxanthineguanine phosphoribosyl transferase.)
For therapeutic use in humans, monoclonal antibodies produced by murine cell lines can be humanized. This is accomplished by attaching the CDRs (the sites that bind antigens) onto appropriate sites in a human immunoglobulin molecule. This produces an antibody that is very similar to a human antibody whose reduced immunogenicity markedly diminishes the chances of an anaphylactic reaction.