CHAPTER OUTLINE

The human immune system monitors the internal and external environment at a molecular level and relays information within itself and to other systems regarding the molecular nature of the environment. Within this process, the system has evolved so that certain stimuli elicit a reaction (an immune response) while others are tolerated, eliciting no response. Microbes that invade our bodies can replicate every 20 min, giving them enormous potential to evolve more virulence. Humans reproduce approximately every 20 years with considerably less potential for population adaptation. However, an immune system that uses overlapping and interwoven processes, and can evolve by somatic mutation, allows an individual to adapt to a changing microbial environment. The immune response, like any other biological process, attempts to restore homoeostasis but may itself, if inappropriate or persistent, result in tissue damage and disease.

Over the last 20 years, there has been a rapid development of our knowledge of immunology, particularly at the biochemical and molecular level, in terms of the sophisticated intercellular interactions that accompany any immune response. This chapter will make no attempt to summarize this vast body of knowledge but rather aims to convey broad concepts in describing immune responses, the immune system that makes the responses and the relevance of these to clinical laboratory medicine. The spectrum of immunological diseases includes genetic deficiencies, autoimmune diseases, allergy, malignancy, inflammation and infection. Manipulation of immune responses by inducing immune activation or suppression has great importance in many areas, including immunization and transplantation.

There are two major types of immune response: innate and adaptive. The innate immune response shares many components with the adaptive response but differs fundamentally in that no specific antigen recognition is required. Instead, it involves recognition of molecular sequences that are widely expressed in nature, for example on microbial cells (lipopolysaccharide) and on damaged tissue (heat shock proteins).

The adaptive response is antigen driven and requires prior antigen exposure and recognition of the antigen by T and B lymphocytes through cell surface receptors. On first contact with any antigen, the system reacts relatively slowly and transient infection may occur. Immunologically, this process is termed immunization. The initial response is known as the primary immune response: it also involves acquisition of memory of the exposure. As a result, on second contact with an antigen there is a more rapid response (the secondary immune response) and there is no infection: the host has acquired immunity. These responses involve a combination of cells, tissues and soluble mediators that are described more fully below. In order to understand immune responses, it is first necessary to consider two important terms: antigen and clonality.

Most biological materials, including proteins, polysaccharides, glycoproteins and polypeptides can be antigens. How the antigen influences the immune system is related to its reaction with lymphocytes, which will determine whether it stimulates an immune response (i.e. it is an immunogen) or is tolerated (i.e. it is a tolerogen). For example, human albumin is tolerated by the human immune system but on injection into a rabbit, it will stimulate an immune response such that the rabbit produces antibodies to the protein. These antibodies, which react with human albumin, but not, for example, with horse albumin or with the rabbit’s own albumin, are described as being specific for human albumin.

The antibodies produced are not directed against the entire molecule but to short, overlapping amino acid sequences termed epitopes. A large protein molecule such as albumin will have many hundreds of epitopes and, generally, the larger the protein, the more immunogenic it is.

Both T and B lymphocytes have the property of antigen recognition through molecules expressed on their cell surfaces (antigen receptors). This is immunoglobulin (Ig) on B cells and the T cell receptor (TCR) on T cells. On any one cell, all the antigen receptors will be identical. Generally, this unique receptor is able to recognize one or a few epitopes. It is estimated that approximately 10¹⁰ different antigenic specificities may potentially be encountered during the lifetime of an individual, requiring approximately this number of unique receptors. The development of this very large number of lymphocyte receptors occurs before antigen contact by random rearrangements of the immunoglobulin and TCR genes. The molecular process by which this is achieved will be described in more detail in a later section. Immune responses are driven by the presence of antigen. Engagement of a compatible receptor with antigen provides signals for further proliferation and differentiation. The affinity of the antigen-receptor binding is crucial in determining the nature of that immune response. This is the molecular basis for the establishment of immune responses and immunological memory.

Additionally, in the B cell, the DNA that codes for the part of the Ig receptor that binds antigen is hypermutable, so that during proliferation, somatic mutations within the receptor DNA sequences occur frequently. These mutations may result in a receptor that has a higher affinity for the antigen and, as a result, the clone bearing that receptor will compete more successfully for the antigen and receive a stronger signal, so that it is preferentially selected and responds more quickly. The secreted immunoglobulin from the B cell will similarly show this higher affinity. This process is termed affinity maturation. Immunity to many potentially lethal or harmful human diseases, for example measles, polio and smallpox, is mediated by such high-affinity immunoglobulin (antibody).

One of the major roles of the immune system is to neutralize and destroy invading pathogens. These pathogens – bacteria, viruses, fungi, protozoans and worms – vary in their size, typical route of entry into the body and mechanism for evading the immune system. Generally, they enter the body across the mucosal surfaces of the respiratory system and gut and across the skin; it is in these areas of the body where the innate immune system forms the first-line of defence from invading pathogens. The major components of the innate immune system are:

• sebaceous secretions; contents include fatty and other acids (hence skin acidity)

• lysozyme (in tears)

• gut acidity

• urine acidity

• intestinal motility

• IGA (in tears, at mucosal surfaces)

• mucus

• ciliated surfaces

• normal bacterial flora

• acute phase proteins

• interferons

• other molecules (including defensins, lactoferrin, peroxidases)

• neutrophils and complement, although these overlap with the acquired immune system.

Intact mucosal surfaces give a high degree of protection against pathogens, and if breached, for example by surgery, burns or mechanical damage, significant infection can occur. The skin is relatively dry with a high salt content; sweat and sebaceous secretions contain fatty acids, triglycerides, lactic acid, amino acids and ammonia that have antimicrobial activity. Mucus and the cilia of the respiratory tract act together to trap and sweep inhaled particles towards the mouth and nose, where they can leave the body or where they can be swallowed to be inactivated by the gastric acid. The gastrointestinal tract has a combination of innate defence mechanisms such as the alkalinity of saliva and the strongly acidic conditions and enzymic activity of the stomach. Mucopolysaccharides in the secretions of the genital tract help make it inhospitable to pathogens. The eyes are bathed in tears containing the enzyme lysozyme, and the rate of tear production can increase to flush away any potential invading pathogens. Similarly, the urinary tract has a high flow rate that helps to keep it free from pathogens.

Many areas of the body are colonized with commensal organisms. It is estimated that a normal gut is colonized with 10¹⁴ bacteria, which form the normal (commensal) bacterial flora and produce chemicals that are antimicrobial and also compete with potentially pathogenetic organisms for essential nutrients. Commensal organisms may cause infection – usually called opportunistic infections – particularly if they get into areas where they do not normally live or if the immune system is compromised.

An adaptive immune response involves an integrated system, in which there are three elements.

2. Cells with specific functions, including specific antigen recognition and presentation.

3. Soluble mediators that participate in immune reactions and can interact with other systems of the body.

Although specific immune responses are made within lymphoid tissues, other non-lymphoid organs have a role to play; for example, the liver is the major site of production of the complement proteins and acute phase proteins.

Neutrophils, in common with basophils and eosinophils, can be shown to contain a large number of cytoplasmic granules when stained and examined by light microscopy and are thus termed granulocytes. They have a multilobed nucleus (polymorphonuclear or PMN, hence their alternative name, polymorphs) and are the most abundant white cells in the circulation. They show high expression of adhesion molecules that enable them to roll along blood vessel walls. If expression of adhesion molecules on the vascular surface is upregulated, for example because of tissue damage or infection, neutrophils are attracted to the site of inflammation (by chemotactic factors) and leave the blood stream and enter the tissues to engulf the invading pathogens or damaged tissue components, and destroy them, in a process termed phagocytosis.

The stages in phagocytosis are as follows.

2. At the site of injury, the particle or microorganism, coated with antibody and complement, adheres to the phagocyte via C3b and F_cγ receptors.

3. The adherence of the particle to the phagocyte results in the activation of the phagocyte’s membrane, initiating a change in its shape and leading to internalization of the foreign material (phagocytosis: literally, ‘eating’ the particle).

4. As the particle is taken into the cell, it becomes surrounded with a membrane, forming a phagocytic vesicle or phagosome.

5. The phagosome fuses with lysosomes to form a phagolysosome, in which the microorganism is killed, and it, or other ingested material, is digested.

6. Completely digested material is reused by the cell; incompletely digested material is released (sometimes accompanied by potentially harmful enzymes).

If phagocytic cells are incubated in vitro with, for example, bacteria, phagocytosis will occur, but the process is significantly enhanced if the bacteria are coated with complement or antibody. This coating is called opsonization and, in simple terms, it enables phagocytic cells to adhere to the marked particles (giving the process specificity) and enables them to change shape to engulf the particles. Many cell types are capable of phagocytosis, the ‘professional’ phagocytic cells being neutrophils and cells of the monocyte/macrophage lineage. Many macrophages home to particular tissues where they develop highly specialized functions, e.g. Kupffer cells (in the liver), mesangial cells (kidneys), microglia (brain) and Langerhans cells (epidermis).

These two cell types are also granulocytes and are particularly important in defending against multicellular pathogens, for example parasitic worms. This defence mechanism relies on the mediators in the cytoplasmic granules rather than the phagocytic functions of the cells. A close relation of the basophil, the mast cell, is tissue-based and important in allergic responses.

Monocytes are immature macrophages that are travelling from their site of production in the bone marrow through the bloodstream to the tissues where they will become fixed macrophages. Potentially, they are phagocytic cells but they do not seem to act as phagocytes in the bloodstream. They have receptors that recognize many bacterial products so are very effective cells in innate immunity. Engagement of these receptors stimulates release of proinflammatory cytokines such as interleukin 1 (IL-1), IL-6 and tumour necrosis factors (TNFs). These cytokines are able to drive many aspects of inflammation and specific immunity. Monocytes therefore provide a valuable second-line defence mechanism once infectious agents have breached the mucosal defences and entered the bloodstream.

The class (or subclass) of the heavy chains and type of light chains is determined by the common amino acid sequences in areas of the molecule called the constant domains. The light chains have one constant domain and the heavy chains have three or four constant domains, depending upon the class; it is these areas that confer functionality to secreted immunoglobulin molecules. Each chain also has a single domain of great variability in the amino acid structure, called the variable domain. The heavy and light chains are arranged such that their variable domains are adjacent, forming a groove in which antigen binding occurs.

If every antigen recognition receptor on B and T cells were encoded on a one gene/one enzyme principle, these receptors alone would require more than the total DNA present within individual cells. The enormous scope for receptors to recognize antigens is achieved by a mechanism of genetic recombination.

IgA and IgM both occur as oligomers of the basic unit. Combination is facilitated by the addition of a short polypeptide chain called the J-chain. This J-chain should not be confused with the J gene segments coding for part of the variable region of the immunoglobulin molecule – the two bear no relationship. In the plasma, IgM occurs as a pentamer. IgA can occur as a dimer and this is the predominant form in secretions. A glycoprotein called ‘secretory piece’ is synthesized by mucosal epithelial cells and integrated into secreted IgA molecules, making them more resistant to degradation by the mucosal environment.

Under normal circumstances, there is enormous microheterogeneity within immunoglobulin molecules. This results in polyclonal immunoglobulins, representing all classes, light chain types and a large number of different idiotypes, each produced by a different B cell clone, even to a single antigen. Monoclonal immunoglobulins, which consist of a single heavy chain class, light chain type and idiotype, result from the proliferation of a single clone of B cells. In fact, polyclonal immunoglobulins comprise very small amounts of many thousands of monoclonal immunoglobulins.

Each class and subclass of immunoglobulin has its own function or group of functions. IgG is the most abundant antibody in the circulation and also diffuses into the extravascular compartment bathing all the tissues. It is produced in secondary immune responses and is vital for immunological memory. There are four subclasses, with IgG1, IgG2, IgG3 and IgG4 being approximately 65%, 25%, 7% and 3%, respectively, of the total IgG. IgG1 and IgG3 responses mature earliest, while IgG2 and IgG4 responses mature more slowly. IgG1, IgG2 and IgG3 are all potent activators of the classical complement cascade. IgG (bound to an antigen) binds to the F_cγ receptors I, II and III on neutrophils, macrophages etc. to facilitate and enhance phagocytosis. IgG has a half-life of approximately 21 days, but this depends upon the plasma concentration, catabolism being increased with raised IgG concentrations and decreased with low IgG concentrations.

IgA is the major immunoglobulin in mucosal secretions and is also produced in secondary immune responses. It is most important in binding and neutralizing organisms at mucosal surfaces, without interacting with other immunological systems.

IgM is the antibody of the primary immune response, culminating in the process of class switching, inducing the B cells to make the antibodies of the secondary responses, IgG or IgA. The large pentameric structure of IgM gives good potential for antigen binding and its high molecular weight keeps it restricted to the vascular compartment. The half-lives of IgA and IgM are independent of their plasma concentrations and are approximately five days for both.

IgD is detectable on B cells early in their development, but its concentration in plasma is normally very low. Its exact function in not known, but it is likely that it is important in processing antigens in immature B cells.

The plasma concentration of IgE is very low; it relies on its binding to the F_cε receptor on mast cells and basophils for its activity. The release of inflammatory mediators within these cells is precipitated by binding of the surface-bound IgE to a specific antigen or allergen.

The T cell receptor is somewhat simpler. It is a heterodimer in which each polypeptide chain is composed of two domains, one variable and one constant. There are four possible polypeptide chains, α, β, γ and δ. A receptor is made up of a pairing of either α with β or γ with δ. In no cell are both types expressed, nor are other pairings possible (e.g. α with δ). The variable domains are similarly constructed from gene segments, as described for immunoglobulin receptors (V, D and J for β and δ; V and J for α and γ), and then spliced into the corresponding constant regions.

The T cell receptor differs from the immunoglobulin receptor in that it has no hypermutable sequences and therefore does not show affinity maturation. Also, it can only recognize antigen that is bound to an HLA (human leukocyte antigen) molecule expressed on the surface of an appropriate antigen presenting cell (APC).

HLA class I molecules are widely expressed on most nucleated cells. They comprise a small non-membrane polypeptide, β₂-microglobulin (the invariant chain of MHC class I) covalently linked to a heavy chain, an integral membrane protein with external domains. The variation within MHC class I arises from polymorphisms within genes for the heavy chains. There are three loci for class I (HLA-A, HLA-B and HLA-C). Each individual inherits A, B and C from each parent and, unlike the situation with immunoglobulins and the TCR, there is no allelic exclusion: both sets of genes are translated. All types occur on any one cell. Because of the polymorphisms, it is likely that any individual will be heterozygous at each locus; therefore six products will be formed.

HLA class II molecules are primarily expressed on immune cells, particularly those able to present antigen. They may be inducible on many other cell types. They comprise two transmembrane polypeptides (α and β chains) of similar size. As for class I, there are three loci (HLA-DR, HLA-DQ and HLA-DP). Again, the likelihood is that these properties will result in at least six distinct receptors. The class II system is, however, more complex. During evolution, there has been replication of some loci such that it is possible to express a number of different DR and DQ types, leading to increased diversity. Even with the variations described above, it is apparent that the total number of different HLA types expressed in any one individual is relatively small compared with immunoglobulins and TCRs.

Unlike immunoglobulin and the TCR, HLA molecules are loaded with peptide antigen intracellularly during molecule assembly, so that prior ‘loaded’ HLA is expressed on the cell surface. Indeed, it would appear that antigen-binding is essential for stability of the molecule and that HLA without antigen is intrinsically unstable. Antigen binds in grooves formed by the folding of class I molecules and the juncture of the α and β chains in class II. The source of the antigen is different for each class. Class I molecules bind antigens synthesized intracellularly, e.g. self-peptides or viral antigens; if these antigens are able to stimulate an immune response it is via CD8 + T cells, resulting in destruction of the target cell. Class II molecules bind antigen that was derived extracellularly, e.g. microbes or microbial products. These antigens are internalized within the APC, where they are subjected to partial proteolytic degradation and then bound to the class II molecules for antigen presentation. These antigens are recognized by CD4 + T cells and stimulate an immune response.

B cells can act as antigen presenting cells. Their surface immunoglobulins are able to react with free antigen molecules, forming complexes that are taken into the cells, where the antigen is degraded into peptides. These peptides can combine with HLA class II molecules to allow antigen presentation to T cells.

Antigen binding alone will rarely stimulate an immune response. Some very repetitive antigen sequences, for example polysaccharides, may directly stimulate B cells. To respond to most antigens, however, both T and B cells require additional signaling from cytokines or contact with other cell types mediated through accessory or co-ligatory molecules expressed on these cells. Accessory cell interactions are many and complex and the reader is referred to dedicated immunological texts for an account of these.

The larger fragment formed on activation usually has the alphabetical annotation ‘b’ (e.g. C3b) and the smaller fragment, ‘a’ (e.g. C3a). Some authors use a line over a component name (e.g. C1) to denote an active enzyme that can activate the next component; this convention is unnecessary and has not been adopted in this chapter.

The alternative pathway is initiated at the level of C3, bypassing the earlier components C1, C4 and C2, which are activated in the classical pathway. C3 spontaneously degrades in tissue fluids and is rapidly inactivated. However, on activating surfaces, including damaged tissue and components of bacterial cell walls such as lipopolysaccharides, degraded C3 is stabilized and can form an enzyme to activate further C3 and to generate an enzyme to cleave C5 and thus activate the terminal sequence of the pathway.

Complexes of antigens with their antibodies (immune complexes) binding C1 activate the classical pathway. C1 is a trimolecular complex of C1s, C1r and C1q. Binding of C1 liberates an enzyme C1s (the C1 esterase), which is able to cleave C4 and C2, and an enzyme formed from a complex of the larger fragments of these two molecules can activate C3. The pathway beyond this is common with the alternative pathway.

Various components of bacterial cell walls are able to bind to a protein in the blood called mannose binding lectin (MBL); this complex is able to stimulate serine proteases that can directly activate component C4.

Many cytokines interact with each other and the effect of a cytokine may change depending upon the prevailing micro-environment. It is important to stress that cytokine synthesis, including that of the proinflammatory cytokines, is a normal response to injury. Their functions should not be thought of as exclusively harmful, resulting in tissue damage or systemic shock. In the majority of instances, cytokines act to coordinate the elimination of invading organisms or removal of damaged tissue, thus avoiding excessive stimulation of specific immunity that could lead to hypersensitivity reactions.

The cytokines interleukin (IL)-1β, IL-6 and tumour necrosis factor alpha (TNFα) are all central to the initiation of the inflammatory response. However, they do not function in isolation: other cytokines implicated in the inflammatory response include IL-8, 10, 11, 12, 17, 18, 23 and interferon γ. In addition, both defensive and injurious effects will involve the release of many mediators of inflammation such as peptides, for example the complement fragment C5a, and lipids, for example platelet activating factor. Many of these mediators act synergistically and also induce each other’s production and the production of other cytokines, resulting in both positive and negative feedback control pathways. Cytokines are also responsible, either directly or indirectly, for healing and successful resolution of the inflammatory insult. Severe inflammatory responses frequently overlap with specific immune responses, as they are often induced by infection and, furthermore, the tissue damage accompanying severe trauma may lead to secondary infection.

Immune responses are usually well controlled and host damage is minimal and reversible. Under some circumstances, resolution does not occur and an exaggerated or persistent response leads to irreversible host tissue damage and, in extreme situations, even death. These inappropriate reactions are termed hypersensitivity reactions. These are classified into four types (I-IV), each with a different mechanism. Type I hypersensitivity reactions, central to allergic responses, may occur without involvement of any other type of hypersensitivity reaction. Most reactions, however, involve more than one type of response and do not, alone, define a pathogenetic mechanism that underlies a group of diseases. There are also other reactions that do not fit neatly into this classification, for example stimulatory reactions, such as when specific immune cells are stimulated by external agents, and when autoantibodies have a stimulatory effect, for example as occurs with thyroid receptor antibodies in Graves disease.

Hypersensitivity reactions result in the release of inflammatory mediators. Some mediators have direct effects on local, or even distant, tissues; others recruit and activate effector cells that further contribute to tissue damage.

The actions of histamine depend on the site of release. In the airways, it induces smooth muscle contraction; in the skin, it causes the hallmark wheal and flare response. Widespread activation of mast cells leads to systemic effects of circulatory shock, hypotension, collapse, chest tightness and, in the most severe cases, respiratory arrest and death: this is anaphylactic shock. Type I hypersensitivity reactions occur rapidly (within approximately 20 min of an insult) and are also called ‘immediate hypersensitivity reactions’.

The important characteristic of type II hypersensitivity reactions is that the antigens involved are localized to the membranes of the target cells; they may be synthesized cell products, cell membrane components or bound foreign molecules (e.g. a drug). The circulating antibodies (IgG or IgM) bind to the cell-bound antigen and activate complement. Full activation of the complement pathway results in the membrane attack complex (C5–9) being assembled on the cell surface, resulting in target cell lysis. Partial activation via C3 will also render the cell a target for phagocytosis, via C3b and IgG that bind to appropriate receptors on phagocytic cells. Activation of phagocytic cells leads to release of further tissue-damaging enzymes. The tissue damage persists for as long as antibody and antigen are present.

Some of the more important type II responses involve red blood cell antigens (including transfusion reactions and haemolytic disease of the newborn) and autoantibodies to cell surface components, for example autoimmune haemolytic anaemia (in which the target is red blood cells), idiopathic thrombocytopenia purpura (in which the target is platelets) and Goodpasture syndrome (in which the target is the kidney glomerular basement membrane).