MODERN DIAGNOSTICS OF DRUG ALLERGY
THERAPEUTIC DRUG MONITORING
Immunology is a relatively new science. Its origin is usually attributed to Edward Jenner, who discovered in 1796 that cowpox, or vaccinia, induced protection against human smallpox, an often fatal disease. Jenner called his procedure vaccination, and this term is still used to describe the inoculation of healthy individuals with weakened or attenuated strains of disease-causing agents to provide protection from disease. Although Jenner's bold experiment was successful, it took almost two centuries for smallpox vaccination to become universal, an advance that enabled the World Health Organization to announce in 1979 that smallpox had been eradicated (Fig. 1.1), arguably the greatest triumph of modern medicine.
The eradication of smallpox by vaccination.
After a period of 3 years in which no cases of smallpox were recorded, the World Health Organization was able to announce in 1979 that smallpox had been eradicated.
When Jenner introduced vaccination he knew nothing of the infectious agents that cause disease: it was not until late in the 19th century that Robert Koch proved that infectious diseases are caused by microorganisms, each one responsible for a particular disease, or pathology. We now recognize four broad categories of disease-causing microorganisms, or pathogens: these are viruses, bacteria, pathogenic fungi, and other relatively large and complex eukaryotic organisms collectively termed parasites. The discoveries of Koch and other great 19th century microbiologists stimulated the extension of Jenner's strategy of vaccination to other diseases. In the 1880s, Louis Pasteur devised a vaccine against cholera in chickens, and developed a rabies vaccine that proved a spectacular success upon its first trial in a boy bitten by a rabid dog. These practical triumphs led to a search for the mechanism of protection and to the development of the science of immunology. In 1890, Emil von Behring and Shibasaburo Kitasato discovered that the serum of vaccinated individuals contained substances which they called antibodies that specifically bound to the relevant pathogen. A specific immune response, such as the production of antibodies against a particular pathogen, is known as an adaptive immune response, because it occurs during the lifetime of an individual as an adaptation to infection with that pathogen. In many cases, an adaptive immune response confers lifelong protective immunity to reinfection with the same pathogen. This distinguishes such responses from innate immunity, which, at the time that von Behring and Kitasato discovered antibodies, was known chiefly through the work of the great Russian immunologist Elie Metchnikoff. Metchnikoff discovered that many microorganisms could be engulfed and digested by phagocytic cells, which he called macrophages. These cells are immediately available to combat a wide range of pathogens without requiring prior exposure and are a key component of the innate immune system. Antibodies, by contrast, are produced only after infection, and are specific for the infecting pathogen. The antibodies present in a given person therefore directly reflect the infections to which he or she has been exposed. Indeed, it quickly became clear that specific antibodies can be induced against a vast range of substances. Such substances are known as antigens because they can stimulate the generation of antibodies. We shall see, however, that not all adaptive immune responses entail the production of antibodies, and the term antigen is now used in a broader sense to describe any substance that can be recognized by the adaptive immune system. Both innate immunity and adaptive immune responses depend upon the activities of white blood cells, or leukocytes. Innate immunity largely involves granulocytes and macrophages. Granulocytes, also called polymorphonuclear leukocytes, are a diverse collection of white blood cells whose prominent granules give them their characteristic staining patterns; they include the neutrophils, which are phagocytic. The macrophages of humans and other vertebrates are presumed to be the direct evolutionary descendants of the phagocytic cells present in simpler animals, such as those that Metchnikoff observed in sea stars. Adaptive immune responses depend upon lymphocytes, which
provide the lifelong immunity that can follow exposure to disease or vaccination. The innate and adaptive immune systems together provide a remarkably effective defense system. It ensures that although we spend our lives surrounded by potentially pathogenic microorganisms, we become ill only relatively rarely. Many infections are
handled successfully by the innate immune system and cause no disease; others that cannot be resolved by innate immunity trigger adaptive immunity and are then overcome successfully, followed by lasting immunological memory.
The components of the immune system.
The cells of the immune system originate in the bone marrow, where many of them also mature. They then migrate to guard the peripheral tissues, circulating in the blood and in a specialized system of vessels called the lymphatic system.
Structure of the immune system.
The white blood cells of the immune system derive from precursors in the bone marrow.
All the cellular elements of blood, including the red blood cells that transport oxygen, the platelets that trigger blood clotting in damaged tissues, and the white blood cells of the immune system, derive ultimately from the same progenitor or precursor cells the hematopoietic stem cells in the bone marrow. As these stem cells can give rise to
all of the different types of blood cells, they are often known as pluripotent hematopoietic stem cells. Initially, they give rise to stem cells of more limited potential, which are the immediate progenitors of red blood cells, platelets, and
the two main categories of white blood cells. The different types of blood cell and their lineage relationships are summarized in Fig. 1.3. We shall be concerned here with all the cells derived from the common lymphoid progenitor and the myeloid progenitor, apart from the megakaryocytes and red blood cells.
All the cellular elements of blood, including the lymphocytes of the adaptive immune system, arise
from hematopoietic stem cells in the bone marrow.
These pluripotent cells divide to produce two more specialized types of stem cells, a common lymphoid progenitor that gives rise to the T and B lymphocytes responsible for adaptive immunity, and a common myeloid progenitor that gives rise to different types of leukocytes (white blood cells), erythrocytes (red blood cells that carry oxygen), and the megakaryocytes that produce platelets that are important in blood clotting. The existence of a common lymphoid progenitor for T and B lymphocytes is strongly supported by current data. T and B lymphocytes are distinguished by their sites of differentiation T cells in the thymus and B cells in the bone marrow and by their antigen receptors. Mature T and B lymphocytes circulate between the blood and peripheral lymphoid tissues. After encounter with antigen, B cells differentiate into antibodysecreting plasma cells, whereas T cells differentiate into effector T cells with a variety of functions. A third lineage of lymphoid-like cells, the natural killer cells, derive from the same progenitor cell but lack the antigen-specificity that is the hallmark of the adaptive immune response (not shown). The leukocytes that derive from the myeloid stem cell are the monocytes, the dendritic cells, and the basophils, eosinophils, and neutrophils. The latter three are collectively termed either granulocytes, because of the cytoplasmic granules whose characteristic staining gives them a distinctive appearance in blood smears, or polymorphonuclear leukocytes, because of their irregularly shaped nuclei. They circulate in the blood and enter the tissues only when recruited to sites of infection or inflammation where neutrophils
are recruited to phagocytose bacteria. Eosinophils and basophils are recruited to sites of allergic inflammation, and appear to be involved in defending against parasites. Immature dendritic cells travel via the blood to enter peripheral tissues, where they ingest antigens. When they encounter a pathogen, they mature and migrate to lymphoid tissues, where they activate antigen-specific T lymphocytes. Monocytes enter tissues, where they differentiate into macrophages; these are the main tissue-resident phagocytic cells of the innate immune system. Mast cells arise from
precursors in bone marrow but complete their maturation in tissues; they are important in allergic responses.
The myeloid progenitor is the precursor of the granulocytes, macrophages, dendritic cells, and mast cells of the immune system. Macrophages are one of the three types of phagocyte in the immune system and are distributed widely in the body tissues, where they play a critical part in innate immunity. They are the mature form of
monocytes, which circulate in the blood and differentiate continuously into macrophages upon migration into the tissues. Dendritic cells are specialized to take up antigen and display it for recognition by lymphocytes. Immature dendritic cells migrate from the blood to reside in the tissues and are both phagocytic and macropinocytic, ingesting large amounts of the surrounding extracellular fluid. Upon encountering a pathogen, they rapidly mature and migrate to lymph nodes.
Mast cells, whose blood-borne precursors are not well defined, also differentiate in the tissues. They mainly reside near small blood vessels and, when activated, release substances that affect vascular permeability. Although best known for their role in orchestrating allergic responses, they are believed to play a part in protecting mucosal surfaces against pathogens. The granulocytes are so called because they have densely staining granules in their cytoplasm; they are also sometimes called polymorphonuclear leukocytes because of their oddly shaped nuclei. There are three types of granulocyte, all of which are relatively short lived and are produced in increased numbers during immune responses, when they leave the blood to migrate to sites of infection or inflammation. Neutrophils, which are the third phagocytic cell of the immune system, are the most numerous and most important cellular component of the innate immune response: hereditary deficiencies in neutrophil function lead to overwhelming bacterial infection, which is fatal if untreated. Eosinophils are thought to be important chiefly in defense against parasitic infections, because their numbers increase during a parasitic infection. The function of basophils is probably similar and complementary to that of eosinophils and mast cells; we shall discuss the functions of these cells and their role in allergic inflammation. The cells of the myeloid lineage are shown
Myeloid cells in innate and adaptive immunity.
Cells of the myeloid lineage perform various important functions in the immune response. The cells are shown schematically in the left column in the form in which they will be represented throughout the rest of the book. A photomicrograph of each cell type is shown in the center column. Macro-phages and neutrophils are primarily phagocytic cells that engulf pathogens and destroy them in intracellular vesicles, a function they perform in both innate and adaptive immune responses. Dendritic cells are phagocytic when they are immature and take up pathogens; after maturing they act as antigen-presenting cells to T cells, initiating adaptive immune responses. Macrophages can also present antigens to T cells and can activate them. The other
myeloid cells are primarily secretory cells that release the contents of their prominent granules upon activation via antibody during an adaptive immune response. Eosinophils are thought to be involved in attacking large antibodycoated parasites such as worms, whereas the function of basophils is less clear. Mast cells are tissue cells that trigger a local inflammatory response to antigen by releasing substances that act on local blood vessels. There are two major types of lymphocyte: B lymphocytes or B cells, which when activated differentiate into plasma cells that secrete antibodies; and T lymphocytes or T cells, of which there are two main classes. One class differentiates on activation into cytotoxic T cells, which kill cells infected with viruses, whereas the second class of T cells differentiates into cells that activate other cells such as B cells and macrophages. Most lymphocytes are small, featureless cells with few cytoplasmic organelles and much of the nuclear chromatin inactive, as shown by its condensed state (Fig. 1.5). This appearance is typical of inactive cells and it is not surprising that, as recently as the early 1960s, textbooks could describe these cells, now the central focus of immunology, as having no known function. Indeed, these small lymphocytes have no functional activity until they encounter antigen, which is necessary to trigger their proliferation and the differentiation of their specialized functional characteristics.
Lymphocytes are mostly small and inactive cells.
The left panel shows a light micrograph of a small lymphocyte surrounded by red blood cells. Note the condensed chromatin of the nucleus, indicating little transcriptional activity, the relative absence of cytoplasm, and the small size. The right panel shows a transmission electron micrograph of a small lymphocyte. Note the condensed chromatin, the scanty cytoplasm and the absence of rough endoplasmic reticulum and other evidence of functional activity. Lymphocytes are remarkable in being able to mount a specific immune response against virtually any foreign antigen. This is possible because each individual lymphocyte matures bearing a unique variant of a prototype antigen receptor, so that the population of T and B lymphocytes collectively bear a huge repertoire of receptors that are highly diverse in their antigen-binding sites. The B-cell antigen receptor (BCR) is a membrane-bound form of the antibody that the B cell will secrete after activation and differentiation to plasma cells. Antibody molecules as a class are known as immunoglobulins, usually shortened to Ig, and the antigen receptor of B lymphocytes is therefore also known as
membrane immunoglobulin (mIg). The T-cell antigen receptor (TCR) is related to immunoglobulin but is quite distinct from it, as it is specially adapted to detect antigens derived from foreign proteins or pathogens that have entered into host cells.
A third lineage of lymphoid cells, called natural killer cells, lack antigenspecific receptors and are part of the innate immune system. These cells circulate in the blood as large lymphocytes with distinctive cytotoxic granules (Fig. 1.6). They are able to recognize and kill some abnormal cells, for example some tumor cells and virus-infected cells, and are thought to be important in the innate immune defense against intracellular pathogens.
Natural killer (NK) cells.
These are large granular lymphocyte-like cells with important functions in
innate immunity. Although lacking antigen-specific receptors, they can detect and attack certain virus-infected cells.
Lymphocytes mature in the bone marrow or the thymus.
The lymphoid organs are organized tissues containing large numbers of lymphocytes in a framework of nonlymphoid cells. In these organs, the interactions lymphocytes make with nonlymphoid cells are important either to lymphocyte development, to the initiation of adaptive immune responses, or to the sustenance of lymphocytes. Lymphoid organs can be divided broadly into central or primary lymphoid organs, where lymphocytes are generated, and peripheral or secondary lymphoid organs, where adaptive immune responses are initiated and where lymphocytes are maintained.The central lymphoid organs are the bone marrow and the thymus, a large organ in the upper chest; the location of the thymus, and of the other lymphoid organs, is shown schematically.
The distribution of lymphoid tissues in the body.
Lymphocytes arise from stem cells in bone marrow, and differentiate in the central lymphoid organs (yellow), B cells in bone marrow and T cells in the thymus. They
migrate from these tissues and are carried in the bloodstream to the peripheral or secondary lymphoid organs (blue), the lymph nodes, the spleen, and lymphoid tissues associated with mucosa, like the gut-associated tonsils, Peyer's patches, and appendix. The peripheral lymphoid organs are the sites of lymphocyte activation by antigen, and lymphocytes recirculate between the blood and these organs until they encounter antigen. Lymphatics drain extracellular fluid from the peripheral tissues, through the lymph nodes and into the thoracic duct, which empties into the left subclavian vein. This fluid, known as lymph, carries antigen to the lymph nodes and recirculatinglymphocytes from the lymph nodes back into the blood. Lymphoid tissue is also associated with other mucosa such as the bronchial linings (not shown).
Both B and T lymphocytes originate in the bone marrow but only B lymphocytes mature there; T lymphocytes migrate to the thymus to undergo their maturation. Thus B lymphocytes are so-called because they are bone marrow derived, and T lymphocytes because they are thymus derived. Once they have completed their maturation, both types of lymphocyte enter the bloodstream, from which they migrate to the peripheral lymphoid organs.
The peripheral lymphoid organs are specialized to trap antigen, to allow the initiation of adaptive immune
responses, and to provide signals that sustain recirculating lymphocytes.
Pathogens can enter the body by many routes and set up an infection anywhere, but antigen and lymphocytes will eventually encounter each other in the peripheral lymphoid organs the lymph nodes, the spleen, and the mucosal lymphoid tissues. Lymphocytes are continually recirculating through these tissues, to which antigen is
also carried from sites of infection, primarily within macrophages and dendritic cells. Within the lymphoid organs, specialized cells such as mature dendritic cells display the antigen to lymphocytes. The lymph nodes are highly organized lymphoid structures located at the points of convergence of vessels of the
lymphatic system, an extensive system of vessels that collects extracellular fluid from the tissues and returns it to the blood. This extracellular fluid is produced continuously by filtration from the blood, and is called lymph. The vessels
are lymphatic vessels or lymphatics. Afferent lymphatic vessels drain fluid from the tissues and also carry antigen-bearing cells and antigens from infected tissues to the lymph nodes, where they are trapped. In the lymph nodes, B lymphocytes are localized in follicles, with T cells more diffusely distributed in surrounding
paracortical areas, also referred to as T-cell zones. Some of the B-cell follicles include germinal centers, where B cells are undergoing intense proliferation after encountering their specific antigen and their cooperating T cells. B and T lymphocytes are segregated in a similar fashion in the other peripheral lymphoid tissues, and we shall see that this organization promotes the crucial interactions that occur between B and T cells upon encountering antigen.
Organization of a lymph node.
As shown in the diagram on the left, a lymph node consists of an outermost cortex and an inner medulla. The cortex is composed of an outer cortex of B cells organized into lymphoid follicles, and deep, or paracortical, areas made up mainly of T cells and dendritic cells. When an immune response is underway, some of the follicles contain central areas of intense B-cell proliferation called germinal centers and are
known as secondary lymphoid follicles. These reactions are very dramatic, but eventually die out as senescent germinal centers. Lymph draining from the extracellular spaces of the body carries antigens in phagocytic dendritic cells and macrophages from the tissues to the lymph node via the afferent lymphatics. Lymph leaves by the efferent lymphatic in the medulla. The medulla consists of strings of macro-phages and antibody-secreting plasma cells known as the medullary cords. Naive lymphocytes enter the node from the bloodstream through specialized
postcapillary venules (not shown) and leave with the lymph through the efferent lymphatic. The light micrograph shows a section through a lymph node, with prominent follicles containing germinal centers. The spleen is a fist-sized organ just behind the stomach that collects antigen from the blood. It also collects and disposes of senescent red blood cells. Its organization is shown schematically. The bulk of the
spleen is composed of red pulp, which is the site of red blood cell disposal. The lymphocytes surround the arterioles entering the organ, forming areas of white pulp, the inner region of which is divided into a periarteriolar lymphoid sheath (PALS), containing mainly T cells, and a flanking B-cell corona.
Organization of the lymphoid tissues of the spleen.
The schematic at top right shows that the spleen consists of red pulp (pink areas in the top panel), which is a site of red blood cell destruction, interspersed with lymphoid white pulp. An enlargement of a small section of the spleen (center) shows the arrangement of discrete areas of white pulp (yellow and blue) around central arterioles. Lymphocytes and antigen- loaded dendritic cells come together in the periarteriolar lymphoid sheath. Most of the white pulp is shown in transverse section, with two portions in longitudinal section. The bottom two schematics show enlargements of a transverse section (lower left) and longitudinal section (lower right) of white pulp. In each area of white pulp, blood carrying lymphocytes and antigen flows from a trabecular artery into a central arteriole. Cells and antigen then pass into a marginal sinus and drain into a trabecular vein. The marginal sinus is surrounded by a marginal zone of lymphocytes. Within the marginal sinus and surrounding the central arteriole is the periarteriolar lymphoid sheath (PALS), made up of T cells. The follicles consist mainly of B cells; in secondary follicles a germinal center is surrounded by a B-cell corona. The light micrograph at bottom left shows a transverse section of white pulp stained with hematoxylin and eosin. The T
cells of the PALS stain darkly, while the B-cell corona is lightly stained. The unstained cells lying between the B- and T-cell areas represent a germinal center. Although the organization of the spleen is similar to that of a lymph node, antigen enters the spleen from the blood rather than from the lymph. The gut-associated lymphoid tissues (GALT), which include the tonsils, adenoids, and appendix, and specialized structures called Peyer's patches in the small intestine, collect antigen from the epithelial surfaces of the gastrointestinal tract. In Peyer's patches, which are the most important and highly organized of these tissues, the antigen is collected by specialized epithelial cells called multi-fenestrated or M cells. The lymphocytes form a follicle consisting of a large central dome of B lymphocytes surrounded by smaller numbers of T lymphocytes. Similar but more diffuse aggregates of lymphocytes protect the respiratory epithelium, where they are known as bronchial-associated lymphoid tissue (BALT), and other mucosa, where they are known simply as mucosal- associated lymphoid tissue (MALT). Collectively, the mucosal immune system is estimated to contain as many lymphocytes as all the rest of the body, and they form a specialized set of cells obeying somewhat different rules.
Organization of typical gut-associated lymphoid tissue.
As the diagram on the left shows, the bulk of the tissue is B cells, organized in a large and highly active domed follicle. T cells occupy the areas between follicles.
The antigen enters across a specialized epithelium made up of so-called M cells. Although this tissue looks very different from other lymphoid organs, the basic divisions are maintained. The light micrograph shows a section through the gut wall. The dome of gut-associated lymphoid tissue can be seen lying beneath the epithelial tissues. Although very different in appearance, the lymph nodes, spleen, and mucosal-associated lymphoid tissues all share the same basic architecture. Each of these tissues operates on the same principle, trapping antigen from sites of
infection and presenting it to migratory small lymphocytes, thus inducing adaptive immune responses. The peripheral lymphoid tissues also provide sustaining signals to the lymphocytes that do not encounter their specific antigen, so
that they continue to survive and recirculate until they encounter their specific antigen. This is important in maintaining the correct numbers of circulating T and B lymphocytes, and ensures that only those lymphocytes with the potential to respond to foreign antigen are sustained.
Lymphocytes circulate between blood and lymph.
Small B and T lymphocytes that have matured in the bone marrow and thymus but have not yet encountered antigen are referred to as naive lymphocytes. These cells circulate continually from the blood into the peripheral lymphoid tissues, which they enter by squeezing between the cells of capillary walls. They are then returned to the blood via the lymphatic vessels or, in the case of the spleen, return directly to the blood. In the event of an infection, lymphocytes that recognize the infectious agent are arrested in the lymphoid tissue, where they proliferate and differentiate into effector cells capable of combating the infection.
Circulating lymphocytes encounter antigen in peripheral lymphoid organs.
Naive lymphocytes recirculate constantly through peripheral lymphoid tissue, here illustrated as a lymph node behind the knee, a popliteal lymph node. Here, they may encounter their specific antigen, draining from an infected site in the foot.
These are called draining lymph nodes, and are the site at which lymphocytes may become activated by encountering their specific ligand.
When an infection occurs in the periphery, for example, large amounts of antigen are taken up by dendritic cells which then travel from the site of infection through the afferent lymphatic vessels into the draining lymph nodes
In the lymph nodes, these cells display the antigen to recirculating T lymphocytes, which they also help to activate. B cells that encounter antigen as they migrate through the lymph node are also arrested and activated, with the help of some of the activated T cells. Once the antigen-specific lymphocytes have undergone a period of
proliferation and differentiation, they leave the lymph nodes as effector cells through the efferent lymphatic vessel Because they are involved in initiating adaptive immune responses, the peripheral lymphoid tissues are not static structures but vary quite dramatically depending upon whether or not infection is present. The diffuse mucosal
lymphoid tissues may appear in response to infection and then disappear, whereas the architecture of the organized tissues changes in a more defined way during an infection. For example, the B-cell follicles of the lymph nodes expand as B lymphocytes proliferate to form germinal centers, and the entire lymph node enlarges, a phenomenon familiarly known as swollen glands.
Immune responses are mediated by leukocytes, which derive from precursors in the bone marrow. A pluripotent hematopoietic stem cell gives rise to the lymphocytes responsible for adaptive immunity, and also to myeloid lineages that participate in both innate and adaptive immunity. Neutrophils, eosinophils, and basophils are
collectively known as granulocytes; they circulate in the blood unless recruited to act as effector cells at sites of infection and inflammation. Macrophages and mast cells complete their differentiation in the tissues where they act as effector cells in the front line of host defense and initiate inflammation. Macrophages phagocytose bacteria, and recruit other phagocytic cells, the neutrophils, from the blood. Mast cells are exocytic and are thought to orchestrate the defense against parasites as well as triggering allergic inflammation; they recruit eosinophils and basophils, which
are also exocytic. Dendritic cells enter the tissues as immature phagocytes where they specialize in ingesting antigens. These antigen-presenting cells subsequently migrate into lymphoid tissue. There are two major types of lymphocyte: B lymphocytes, which mature in the bone marrow; and T lymphocytes, which mature in the thymus. The bone marrow and thymus are thus known as the central or primary lymphoid organs. Mature lymphocytes recirculate continually from the bloodstream through the peripheral or secondary lymphoid organs, returning to the bloodstream
through the lymphatic vessels. Most adaptive immune responses are triggered when a recirculating T cell recognizes its specific antigen on the surface of a dendritic cell. The three major types of peripheral lymphoid tissue are the spleen, which collects antigens from the blood; the lymph nodes, which collect antigen from sites of infection in the tissues; and the mucosal-associated lymphoid tissues (MALT), which collect antigens from the epithelial surfaces of the body. Adaptive immune responses are initiated in these peripheral lymphoid tissues: T cells that encounter
antigen proliferate and differentiate into antigen-specific effector cells, while B cells proliferate and differentiate into antibody-secreting cells.
Principles of innate and adaptive immunity.
The macrophages and neutrophils of the innate immune system provide a first line of defense against many common microorganisms and are essential for the control of common bacterial infections. However, they cannot always
eliminate infectious organisms, and there are some pathogens that they cannot recognize. The lymphocytes of the adaptive immune system have evolved to provide a more versatile means of defense which, in addition, provides increased protection against subsequent reinfection with the same pathogen. The cells of the innate immune system, however, play a crucial part in the initiation and subsequent direction of adaptive immune responses, as well as participating in the removal of pathogens that have been targeted by an adaptive immune response. Moreover,
because there is a delay of 4-7 days before the initial adaptive immune response takes effect, the innate immune response has a critical role in controlling infections during this period.
Most infectious agents induce inflammatory responses by activating innate immunity.
Microorganisms such as bacteria that penetrate the epithelial surfaces of the body for the first time are met immediately by cells and molecules that can mount an innate immune response. Phagocytic macrophages conduct the defense against bacteria by means of surface receptors that are able to recognize and bind common constituents of many bacterial surfaces. Bacterial molecules binding to these receptors trigger the macrophage to engulf the bacterium and also induce the secretion of biologically active molecules. Activated macrophages secrete cytokines,
which are defined as proteins released by cells that affect the behavior of other cells that bear receptors for them. They also release proteins known as chemokines that attract cells with chemokine receptors such as neutrophils and monocytes from the bloodstream. The cytokines and chemokines released by macrophages in response to
bacterial constituents initiate the process known as inflammation. Local inflammation and the phagocytosis of invading bacteria may also be triggered as a result of the activation of complement on the bacterial cell surface. Complement is a system of plasma proteins that activates a cascade of proteolytic reactions on microbial surfaces but not on host cells, coating these surfaces with fragments that are recognized and bound by phagocytic receptors on macrophages. The cascade of reactions also releases small peptides that contribute to inflammation.
Bacterial infection triggers an inflammatory response.
Macrophages encountering bacteria in the tissues are triggered to release cytokines that increase the permeability of blood vessels, allowing fluid and proteins to
pass into the tissues. They also produce chemokines that direct the migration of neutrophils to the site of infection. The stickiness of the endothelial cells of the blood vessels is also changed, so that cells adhere to the blood vessel wall and are able to crawl through it; first neutrophils and then monocytes are shown entering the tissue from a blood vessel. The accumulation of fluid and cells at the site of infection causes the redness, swelling, heat, and pain, known collectively as inflammation. Neutrophils and macrophages are the principal inflammatory cells. Later in an immune response, activated lymphocytes may also contribute to inflammation.
Inflammation is traditionally defined by the four Latin words calor, dolor, rubor, and tumor, meaning heat, pain, redness, and swelling, all of which reflect the effects of cytokines and other inflammatory mediators on the local blood vessels. Dilation and increased permeability of the blood vessels during inflammation lead to increased local blood flow and the leakage of fluid, and account for the heat, redness, and swelling. Cytokines and complement fragments also have important effects on the adhesive properties of the endothelium, causing circulating leukocytes to
stick to the endothelial cells of the blood vessel wall and migrate between them to the site of infection, to which they are attracted by chemokines. The migration of cells into the tissue and their local actions account for the pain. The main cell types seen in an inflammatory response in its initial phases are neutrophils, which are recruited into the inflamed, infected tissue in large numbers. Like macrophages, they have surface receptors for common bacterial constituents and complement, and they are the principal cells that engulf and destroy the invading micro-organisms.
The influx of neutrophils is followed a short time later by monocytes that rapidly differentiate into macrophages. Macrophages and neutrophils are thus also known as inflammatory cells. Inflammatory responses later in an
infection also involve lymphocytes, which have meanwhile been activated by antigen that has drained from the site of infection via the afferent lymphatics.
The innate immune response makes a crucial contribution to the activation of adaptive immunity. The inflammatory response increases the flow of lymph containing antigen and antigen-bearing cells into lymphoid tissue, while complement fragments on microbial surfaces and induced changes in cells that have taken up microorganisms provide signals that synergize in activating lymphocytes whose receptors bind to specific microbial antigens. Macrophages that have phagocytosed bacteria and become activated can also activate T lymphocytes. However, the
cells that specialize in presenting antigen to T lymphocytes and initiating adaptive immunity are the dendritic cells.
Activation of specialized antigen-presenting cells is a necessary first step for induction of adaptive
The induction of an adaptive immune response begins when a pathogen is ingested by an immature dendritic cell in the infected tissue. These specialized phagocytic cells are resident in most tissues and are relatively long-lived, turning over at a slow rate. They derive from the same bone marrow precursor as macrophages, and migrate from the bone marrow to their peripheral stations, where their role is to survey the local environment for pathogens. Eventually, all tissue-resident dendritic cells migrate through the lymph to the regional lymph nodes where they interact with recirculating naive lymphocytes. If the dendritic cells fail to be activated, they induce tolerance to the antigens of self that they bear. The immature dendritic cell carries receptors on its surface that recognize common features of many pathogens, such as bacterial cell wall proteoglycans. As with macrophages and neutrophils, binding of a bacterium to these receptors stimulates the dendritic cell to engulf the pathogen and degrade it intracellularly. Immature dendritic cells are also continually taking up extracellular material, including any virus particles or bacteria that may be present, by the
receptor-independent mechanism of macropinocytosis. The function of dendritic cells, however, is not primarily to destroy pathogens but to carry pathogen antigens to peripheral lymphoid organs and there present them to T lymphocytes. When a dendritic cell takes up a pathogen in infected tissue, it becomes activated, and travels to a nearby lymph node. On activation, the dendritic cell matures into a highly effective antigen-presenting cell (APC) and undergoes changes that enable it to activate pathogen-specific lymphocytes that it encounters in the lymph node
Activated dendritic cells secrete cytokines that influence both innate and adaptive immune responses, making these cells essential gatekeepers that determine whether and how the immune system responds to the presence of infectious agents.
Dendritic cells initiate adaptive immune responses.
Immature dendritic cells resident in infected tissues take up pathogens and their antigens by macropinocytosis and receptor-mediated phagocytosis. They are
stimulated by recognition of the presence of pathogens to migrate via the lymphatics to regional lymph nodes, where they arrive as fully mature nonphagocytic dendritic cells. Here the mature dendritic cell encounters and activates
antigen-specific naive T lymphocytes, which enter lymph nodes from the blood via a specialized vessel known from its cuboidal endothelial cells as a high endothelial venule (HEV).
Lymphocytes activated by antigen give rise to clones of antigen-specific cells that mediate adaptive
The defense systems of innate immunity are effective in combating many pathogens. They are constrained, however, by relying on germline-encoded receptors to recognize microorganisms that can evolve more rapidly than the hosts they infect. This explains why they can only recognize microorganisms bearing surface molecules that are common to many pathogens and that have been conserved over the course of evolution. Not surprisingly, many pathogenic bacteria have evolved a protective capsule that enables them to conceal these molecules and thereby avoid being recognized and phagocytosed. Viruses carry no invariant molecules similar to those of bacteria and are rarely recognized directly by macrophages. Viruses and encapsulated bacteria can, however, still be taken up by dendritic cells through the nonreceptor-dependent process of macropinocytosis. Molecules that reveal their infectious nature may then be unmasked, and the dendritic cell activated to present their antigens to lymphocytes. The recognition mechanism used by the lymphocytes of the adaptive immune response has evolved to overcome the constraints faced
by the innate immune system, and enables recognition of an almost infinite diversity of antigens, so that each different pathogen can be targeted specifically.
Instead of bearing several different receptors, each recognizing a different surface feature shared by many pathogens, each naive lymphocyte entering the bloodstream bears antigen receptors of a single specificity. The specificity of
these receptors is determined by a unique genetic mechanism that operates during lymphocyte development in the bone marrow and thymus to generate millions of different variants of the genes encoding the receptor molecules. Thus, although an individual lymphocyte carries receptors of only one specificity, the specificity of each lymphocyte is different. This ensures that the millions of lymphocytes in the body collectively carry millions of different antigen receptor specificities the lymphocyte receptor repertoire of the individual. During a person's lifetime these lymphocytes undergo a process akin to natural selection; only those lymphocytes that encounter an antigen to which their receptor binds will be activated to proliferate and differentiate into effector cells.This selective mechanism was first proposed in the 1950s by Macfarlane Burnet to explain why antibodies, which can be induced in response to virtually any antigen, are produced in each individual only to those antigens to which
he or she is exposed. He postulated the preexistence in the body of many different potential antibody-producing cells, each having the ability to make antibody of a different specificity and displaying on its surface a membrane-bound version of the antibody that served as a receptor for antigen. On binding antigen, the cell is activated to divide and produce many identical progeny, known as a clone; these cells can now secrete clonotypic antibodies with a specificity identical to that of the surface receptor that first triggered activation and clonal expansion.
Burnet called this the clonal selection theory.
Each lymphocyte progenitor gives rise to many lymphocytes, each bearing a distinct
antigen receptor. Lymphocytes with receptors that bind ubiquitous self antigens are eliminated before they become fully mature, ensuring tolerance to such self antigens. When antigen interacts with the receptor on a mature naïve lymphocyte, that cell is activated and starts to divide. It gives rise to a clone of identical progeny, all of whose receptors bind the same antigen. Antigen specificity is thus maintained as the progeny proliferate and differentiate into effector cells. Once antigen has been eliminated by these effector cells, the immune response ceases.
Clonal selection of lymphocytes is the central principle of adaptive immunity.
Remarkably, at the time that Burnet formulated his theory, nothing was known of the antigen receptors of lymphocytes; indeed the function of lymphocytes themselves was still obscure. Lymphocytes did not take center stage until the early 1960s, when James Gowans discovered that removal of the small lymphocytes from rats resulted
in the loss of all known adaptive immune responses. These immune responses were restored when the small lymphocytes were replaced. This led to the realization that lymphocytes must be the units of clonal selection, and their biology became the focus of the new field of cellular immunology.
Clonal selection of lymphocytes with diverse receptors elegantly explained adaptive immunity but it raised one significant intellectual problem. If the antigen receptors of lymphocytes are generated randomly during the lifetime of an individual, how are lymphocytes prevented from recognizing antigens on the tissues of the body and attacking them? Ray Owen had shown in the late 1940s that genetically different twin calves with a common placenta were immunologically tolerant of one another's tissues, that is, they did not make an immune response against each other.
Sir Peter Medawar then showed in 1953 that if exposed to foreign tissues during embryonic development, mice become immunologically tolerant to these tissues. Burnet proposed that developing lymphocytes that are potentially
self-reactive are removed before they can mature, a process known as clonal deletion. He has since been proved right in this too, although the mechanisms of tolerance are still being worked out, as we shall see when we discuss the development of lymphocytes.
Clonal selection of lymphocytes is the single most important principle in adaptive immunity. Its four basic postulates are listed. The last of the problems posed by the clonal selection theory how the diversity of lymphocyte antigen receptors is generated was solved in the 1970s when advances in molecular biology made it
possible to clone the genes encoding antibody molecules.
The four basic principles of clonal selection.
The structure of the antibody molecule illustrates the central puzzle of adaptive immunity.
Antibodies, as discussed above, are the secreted form of the B-cell antigen receptor or BCR. Because they are produced in very large quantities in response to antigen, they can be studied by traditional biochemical techniques; indeed, their structure was understood long before recombinant DNA technology made it possible to study the
membrane-bound antigen receptors of lymphocytes. The startling feature that emerged from the biochemical studies was that an antibody molecule is composed of two distinct regions. One is a constant region that can take one of only
four or five biochemically distinguishable forms; the other is a variable region that can take an apparently infinite variety of subtly different forms that allow it to bind specifically to an equally vast variety of different antigens.
This division is illustrated in the simple schematic diagram, where the antibody is depicted as a Y-shaped molecule, with the constant region shown in blue and the variable region in red. The two variable regions, which are
identical in any one antibody molecule, determine the antigen-binding specificity of the antibody; the constant region determines how the antibody disposes of the pathogen once it is bound.
Schematic structure of an antibody molecule.
The two arms of the Y-shaped antibody molecule contain the variable regions that form the two identical antigen-binding sites. The stem can take one of only a limited
number of forms and is known as the constant region. It is the region that engages the effector mechanisms that antibodies activate to eliminate pathogens.
Each antibody molecule has a twofold axis of symmetry and is composed of two identical heavy chains and two identical light chains. Heavy and light chains both have variable and constant regions; the variable regions of a heavy and a light chain combine to form an antigen-binding site, so that both chains contribute to the antigenbinding specificity of the antibody molecule. For the time being we are concerned only with the properties of immunoglobulin molecules as
antigen receptors, and how the diversity of the variable regions is generated.
Antibodies are made up of four protein chains.
There are two types of chain in an antibody molecule:
a larger chain called the heavy chain (green), and a smaller one called the light chain (yellow). Each chain has both a variable and a constant region, and there are two identical light chains and two identical heavy chains in each antibody molecule.
Each developing lymphocyte generates a unique antigen receptor by rearranging its receptor genes.
How are antigen receptors with an almost infinite range of specificities encoded by a finite number of genes? This question was answered in 1976, when Susumu Tonegawa discovered that the genes for immunoglobulin variable regions are inherited as sets of gene segments, each encoding a part of the variable region of one of the immunoglobulin polypeptide chains. During B-cell development in the bone marrow, these gene segments are irreversibly joined by DNA recombination to form a stretch of DNA encoding a complete variable region. Because there are many different gene segments in each set, and different gene segments are joined together in different cells, each cell generates unique genes for the variable regions of the heavy and light chains of the immunoglobulin molecule. Once these recombination events have succeeded in producing a functional receptor, further rearrangement is prohibited. Thus each lymphocyte expresses only one receptor specificity.
The diversity of lymphocyte antigen receptors is generated by somatic gene rearrangements.
Different parts of the variable regions of antigen receptors are encoded by sets of gene segments. During a lymphocyte's development, one member of each set of gene segments is joined randomly to the others by an irreversible process of DNA recombination. The juxtaposed gene segments make up a complete gene that encodes the variable part of one chain of the receptor, and is unique to that cell. This random rearrangement is repeated for the set of gene segments encoding the other chain. The rearranged genes are expressed to produce the two types of polypeptide chain. These come together to form a unique antigen receptor on the lymphocyte surface. Each
lymphocyte bears many copies of its unique receptor. This mechanism has three important consequences. First, it enables a limited number of gene segments to generate a vast number of different proteins. Second, because each cell assembles a different set of gene segments, each cell expresses a unique receptor specificity. Third, because gene rearrangement involves an irreversible change in a cell's
DNA, all the progeny of that cell will inherit genes encoding the same receptor specificity. This general scheme was later also confirmed for the genes encoding the antigen receptor on T lymphocytes. The main distinctions between B-
and T-lymphocyte receptors are that the immunoglobulin that serves as the B-cell antigen receptor has two identical antigen-recognition sites and can also be secreted, whereas the T-cell antigen receptor has a single antigenrecognition site and is always a cell-surface molecule. We shall see later that these receptors also recognize antigen in very different ways. The potential diversity of lymphocyte receptors generated in this way is enormous. Just a few hundred different gene segments can combine in different ways to generate thousands of different receptor chains. The diversity of
lymphocyte receptors is further amplified by junctional diversity, created by adding or subtracting nucleotides in the process of joining the gene segments, and by the fact that each receptor is made by pairing two different variable chains, each encoded in distinct sets of gene segments. A thousand different chains of each type could thus generate 106 distinct antigen receptors through this combinatorial diversity. Thus a small amount of genetic material can encode a truly staggering diversity of receptors. Only a subset of these randomly generated receptor specificities survive the selective processes that shape the peripheral lymphocyte repertoire; nevertheless, there are lymphocytes of at least 108 different specificities in an individual at any one time. These provide the raw material on which clonal selection acts.
Lymphocyte development and survival are determined by signals received through their antigen
Equally amazing as the generation of millions of specificities of lymphocyte antigen receptors is the shaping of this repertoire during lymphocyte development and the homeostatic maintenance of such an extensive repertoire in the periphery. How are the most useful receptor specificities selected, and how are the numbers of peripheral
lymphocytes, and the percentages of B cells and T cells kept relatively constant? The answer seems to be that lymphocyte maturation and survival are regulated by signals received through their antigen receptors. Strong signals received through the antigen receptor by an immature lymphocyte cause it to die or undergo further receptor
rearrangement, and in this way self-reactive receptor specificities are deleted from the repertoire. However, a complete absence of signals from the antigen receptor can also lead to cell death. It seems that in order to survive, lymphocytes must periodically receive certain signals from their environment via their antigen receptors. In this way,
the body can ensure that each receptor is functional and regulate the number and type of lymphocytes in the population at any given time. These survival signals appear to be delivered by other cells in the lymphoid organs and must derive, at least in part, from the body's own molecules, the self antigens, as altering the self environment alters the life-span of lymphocytes in that environment. Developing B cells in the bone marrow interact with stromal cells, while their final maturation and continued recirculation appears to depend on survival signals received from the Bcell
follicles of peripheral lymphoid tissue. T lymphocytes receive survival signals from self molecules on specialized epithelial cells in the thymus during development, and from the same molecules expressed by dendritic cells in the lymphoid tissues in the periphery. The self ligands that interact with the T-cell receptor to deliver these signals are partially defined, being composed of known cell-surface molecules complexed with undefined peptides from other
self proteins in the cell. These same cell-surface molecules function to present foreign intracellular antigens to T
cells. They select only a subset of T-cell receptors for survival, but these are the receptors most likely to be useful in responding to foreign antigens.
Lymphocytes that fail to receive survival signals, and those that are clonally deleted because they are self-reactive, undergo a form of cell suicide called apoptosis or programmed cell death. Apoptosis, derived from a Greek word meaning the falling of leaves from the trees, occurs in all tissues, at a relatively constant rate in each tissue, and is a means of regulating the number of cells in the body. It is responsible, for example, for the death and shedding of skin cells, the turnover of liver cells, and the death of the oldest intestinal epithelial cells that are constantly replaced by new cells. Thus, it should come as no surprise that immune system cells are regulated through the same mechanism. Each day the bone marrow produces many millions of new neutrophils, monocytes, red blood cells, and lymphocytes, and this production must be balanced by an equal loss of these cells. Regulated loss of all these blood cells occurs by apoptosis, and the dying cells are finally phagocytosed by specialized macrophages in the liver and spleen. Lymphocytes are a special case, because the loss of an individual naive lymphocyte means the loss of a receptor specificity from the repertoire, while each newly matured cell that survives will contribute a different specificity. The survival signals received through the antigen receptors appear to regulate this process by inhibiting the apoptosis of individual lymphocytes, thus regulating the maintenance and composition of the lymphocyte repertoire.
Lymphocytes proliferate in response to antigen in peripheral lymphoid organs, generating effector cells
and immunological memory.
The large diversity of lymphocyte receptors means that there will usually be at least a few that can bind to any given foreign antigen. However, because each lymphocyte has a different receptor, the numbers of lymphocytes that can bind and respond to any given antigen is very small. To generate sufficient antigen-specific effector lymphocytes to fight an infection, a lymphocyte with an appropriate receptor specificity must be activated to proliferate before its progeny finally differentiate into effector cells. This clonal expansion is a feature common to all adaptive immune
responses. As we have seen, lymphocyte activation and proliferation is initiated in the draining lymphoid tissues, where naïve lymphocytes and activated antigen-presenting cells can come together. Antigens are thus presented to the naïve recirculating lymphocytes as they migrate through the lymphoid tissue before returning to the bloodstream via the efferent lymph. On recognizing its specific antigen, a small lymphocyte stops migrating and enlarges. The chromatin in its nucleus becomes less dense, nucleoli appear, the volume of both the nucleus and the cytoplasm increases, and new RNAs and proteins are synthesized. Within a few hours, the cell looks completely different and is known as a lymphoblast.
The lymphoblasts now begin to divide, normally duplicating themselves two to four times every 24 hours for 3 to 5 days, so that one naive lymphocyte gives rise to a clone of around 1000 daughter cells of identical specificity. These then differentiate into effector cells. In the case of B cells, the differentiated effector cells, the plasma
cells, secrete antibody; in the case of T cells, the effector cells are able to destroy infected cells or activate other cells of the immune system. These changes also affect the recirculation of antigen-specific lymphocytes. Changes in the
cell-adhesion molecules they express on their surface allow effector lymphocytes to migrate into sites of infection or stay in the lymphoid organs to activate B cells.
Transmission electron micrographs of lymphocytes at various stages of activation to effector function.
The course of a typical antibody response.
First encounter with an antigen produces a primary response. Antigen A introduced at time zero encounters little specific antibody in the serum. After a lag phase, antibody against antigen A (blue) appears; its concentration rises to a plateau, and then declines. When the serum is tested for antibody against another antigen, B (yellow), there is none present, demonstrating the specificity of the antibody response. When the animal is later challenged with a mixture of antigens A and B, a very rapid and intense secondary response to A occurs. This illustrates immunological memory, the ability of the immune system to make a second response to the same antigen more efficiently and effectively, providing the host with a specific defense against infection. This is the main reason for giving booster injections after an initial vaccination. Note that the response to B resembles the initial or primary response to A, as this is the first encounter of the animal with antigen B.
It is immunological memory that enables successful vaccination and prevents reinfection with pathogens that have been repelled successfully by an adaptive immune response. Immunological memory is the most important biological
consequence of the development of adaptive immunity, although its cellular and molecular basis is still not fully understood.
The professional antigen-presenting cells.
The major pathogen types confronting the immune system and some of the diseases that they cause.
The effector mechanisms that operate to eliminate pathogens in an adaptive immune response are essentially identical to those of innate immunity. Indeed, it seems likely that specific recognition by clonally distributed receptors evolved as a late addition to existing innate effector mechanisms to produce the present-day adaptive immune response. We begin by outlining the effector actions of antibodies, which depend almost entirely on recruiting cells and molecules of the innate immune system.
Antibodies deal with extracellular forms of pathogens and their toxic products.
Antibodies, which were the first specific product of the adaptive immune response to be identified, are found in the fluid component of blood, or plasma, and in extracellular fluids. Because body fluids were once known as humors, immunity mediated by antibodies is known as humoral immunity.
As we have seen, antibodies are Y-shaped molecules whose arms form two identical antigen-binding sites. These are highly variable from one molecule to another, providing the diversity required for specific antigen recognition. The stem of the Y, which defines the class of the antibody and determines its functional properties, takes
one of only five major forms, or isotypes. Each of the five antibody classes engages a distinct set of effector mechanisms for disposing of antigen once it is recognized.
Schematic structure of an antibody molecule.
The two arms of the Y-shaped antibody molecule contain the variable regions that form the two identical antigen-binding sites. The stem can take one of only a limited
number of forms and is known as the constant region. It is the region that engages the effector mechanisms that antibodies activate to eliminate pathogens.
The simplest and most direct way in which antibodies can protect from pathogens or their toxic products is by binding to them and thereby blocking their access to cells that they might infect or destroy (left panels).
This is known as neutralization and is important for protection against bacterial toxins and against pathogens such as viruses, which can thus be prevented from entering cells and replicating.
Binding by antibodies, however, is not sufficient on its own to arrest the replication of bacteria that multiply outside cells. In this case, one role of antibody is to enable a phagocytic cell to ingest and destroy the bacterium. This is important for the many bacteria that mare resistant to direct recognition by phagocytes; instead, the phagocytes recognize the constant region of the antibodies coating the bacterium. The coating of pathogens and foreign particles in this way is known as opsonization.
Antibodies can participate in host defense in three main ways.
The left panels show antibodies binding to and neutralizing a bacterial toxin, thus preventing it from interacting with host cells and causing pathology.
Unbound toxin can react with receptors on the host cell, whereas the toxin:antibody complex cannot. Antibodies also neutralize complete virus particles and bacterial cells by binding to them and inactivating them. The antigen:antibody complex is eventually scavenged and degraded by macrophages. Antibodies coating an antigen render it recognizable as foreign by phagocytes (macrophages and neutrophils), which then ingest and destroy it; this is called opsonization.
The middle panels show opsonization and phagocytosis of a bacterial cell. The right panels show activation of the complement system by antibodies coating a bacterial cell. Bound antibodies form a receptor for the first protein of the complement system, which eventually forms a protein complex on the surface of the bacterium that, in some cases, can kill the bacterium directly. More generally, complement coating favors the uptake and destruction of the bacterium by phagocytes. Thus, antibodies target pathogens and their toxic products for disposal by phagocytes.
The third function of antibodies is to activate a system of plasma proteins known as complement. The complement system, which we shall discuss in detail, can also be activated without the help of antibodies on many microbial surfaces, and therefore contributes to innate as well as adaptive immunity. The pores formed by activated
complement components directly destroy bacteria, and this is important in a few bacterial infections. However, the main function of complement, like that of the antibodies themselves, is to coat the surface of pathogens and enable phagocytes to engulf and destroy bacteria that they would otherwise not recognize.
Complement also enhances the bactericidal actions of phagocytes; indeed it is so-called because it 'complements' the activities of antibodies.
Antibodies of different isotypes are found in different compartments of the body and differ in the effector mechanisms that they recruit, but all pathogens and particles bound by antibody are eventually delivered to phagocytes for ingestion, degradation, and removal from the body. The complement system and the phagocytes that antibodies recruit are not themselves antigen-specific; they depend upon antibody
molecules to mark the particles as foreign. Antibodies are the sole contribution of B cells to the adaptive immune response. T cells, by contrast, have a variety of effector actions.
Antibodies can participate in host defense in three main ways.
The left panels show antibodies binding to and neutralizing a bacterial toxin, thus preventing it from interacting with host cells and causing pathology.
Unbound toxin can react with receptors on the host cell, whereas the toxin:antibody complex cannot. Antibodies also neutralize complete virus particles and bacterial cells by binding to them and inactivating them. The antigen:antibody
complex is eventually scavenged and degraded by macrophages. Antibodies coating an antigen render it recognizable as foreign by phagocytes (macrophages and neutrophils), which then ingest and destroy it; this is called opsonization.
The middle panels show opsonization and phagocytosis of a bacterial cell. The right panels show activation of the complement system by antibodies coating a bacterial cell. Bound antibodies form a receptor for the first protein of the complement system, which eventually forms a protein complex on the surface of the bacterium that, in some cases, can kill the bacterium directly. More generally, complement coating favors the uptake and destruction of the bacterium by phagocytes. Thus, antibodies target pathogens and their toxic products for disposal by phagocytes.
T cells are needed to control intracellular pathogens and to activate B-cell responses to most antigens.
Pathogens are accessible to antibodies only in the blood and the extracellular spaces. However, some bacterial pathogens and parasites, and all viruses, replicate inside cells where they cannot be detected by antibodies. The destruction of these invaders is the function of the T lymphocytes, or T cells, which are responsible for the cellmediated immune responses of adaptive immunity.
Cell-mediated reactions depend on direct interactions between T lymphocytes and cells bearing the antigen that the T cells recognize. The actions of cytotoxic T cells are the most direct. These recognize any of the body's cells that are infected with viruses, which replicate inside cells, using the biosynthetic machinery of the cell itself. The replicating virus eventually kills the cell, releasing new virus particles. Antigens derived from the replicating virus are, however, displayed on the surface of infected cells, where they are recognized by cytotoxic T cells. These cells can then control the infection by killing the infected cell before viral replication is complete. Cytotoxic T cells typically express the molecule CD8 on their cell surfaces.
Mechanism of host defense against intracellular infection by viruses.
Cells infected by viruses are recognized by specialized T cells called cytotoxic T cells, which kill the infected cells directly.
The killing mechanism involves the activation of enzymes known as caspases, which cleave after aspartic acid. These in turn activate a cytostolic nuclease in the infected cell, which cleaves host and viral DNA. Panel a is a transmission electron micrograph showing the plasma membrane of a cultured CHO cell (the Chinese hamster ovary cell line) infected with influenza virus. Many virus particles can be seen budding from the cell surface. Some of these have been labeled with a monoclonal antibody that is specific for a viral protein and is coupled to gold particles, which appear as the solid black dots in the micrograph. Panel b is a transmission electron micrograph of a virus-infected cell (V) surrounded by cytotoxic T lymphocytes. Note the close apposition of the membranes of the virus-infected cell and the T cell (T) in the upper left corner of the micrograph, and the clustering of the cytoplasmic organelles in the T cell between the nucleus and the point of contact with the infected cell.
Other T lymphocytes that activate the cells they recognize are marked by the expression of the cell-surface molecule CD4 instead of CD8. Such T cells are often generically called helper T, or TH cells, but this is a term that we will use
for a specific subset of CD4 T cells. CD4 T lymphocytes can be divided into two subsets, which carry out different functions in defending the body, in particular from bacterial infections. The first subset of CD4 T lymphocytes is important in the control of intracellular bacterial infections.
Some bacteria grow only in the intracellular membranebounded vesicles of macrophages; important examples are Mycobacterium tuberculosis and M. leprae, the pathogens that cause tuberculosis and leprosy. Bacteria phagocytosed by macrophages are usually destroyed in the lysosomes, which contain a variety of enzymes and antimicrobial substances. Intracellular bacteria survive because the vesicles they occupy do not fuse with the lysosomes. These infections can be controlled by a subset of CD4 T cells, known as a TH1 cells, which activate macrophages, inducing the fusion of their lysosomes with the vesicles containing the
bacteria and at the same time stimulating other antibacterial mechanisms of the phagocyte. TH1 cells also release cytokines and chemokines that attract macrophages to the site of infection.
Mechanism of host defense against intracellular infection by mycobacteria.
Mycobacteria are engulfed by macrophages but resist being destroyed by preventing the fusion of the intracellular vesicles in which they reside with the lysosomes containing bactericidal agents; instead they persist and replicate in these vesicles.
However, when a specific TH1 cell recognizes an infected macrophage, it releases cytokines that activate the macrophage and induce lysosomal fusion and macrophage bactericidal activity. The light micrographs (bottom row) show resting (left) and activated (right) macrophages infected with mycobacteria. The cells have been stained with an acid-fast red dye to reveal mycobacteria. These are prominent as red-staining rods in the resting macrophages but have been eliminated from the activated macrophages.
T cells destroy intracellular pathogens by killing infected cells and by activating macrophages but they also have a central role in the destruction of extracellular pathogens by activating B cells. This is the specialized role of the second subset of CD4 T cells, called TH2 cells.
We shall see, when we discuss the humoral immune response in detail, that only a few antigens with special properties can activate naive B lymphocytes on their own.
Most antigens require an accompanying signal from helper T cells before they can stimulate B cells to proliferate and differentiate into cells secreting antibody. The ability of T cells to activate B cells was discovered long before it was recognized that a functionally distinct class of T cells activates macrophages, and the term helper T cell was originally coined to describe T cells that activate B cells. Although the designation 'helper' was later extended to T cells that activate macrophages (hence the H in TH1), we consider this usage confusing and we will, in the remainder of this book, reserve the term helper T cells for all T cells that activate B cells.
T cells are specialized to recognize foreign antigens as peptide fragments bound to proteins of the major histocompatibility complex.
All the effects of T lymphocytes depend upon interactions with target cells containing foreign proteins. Cytotoxic T cells and TH1 cells interact with antigens produced by pathogens that have have infected the target cell or that have been ingested by it. Helper T cells, in contrast, recognize and interact with B cells that have bound and internalized foreign antigen by means of their surface immunoglobulin. In all cases, T cells recognize their targets by detecting peptide fragments derived from the foreign proteins, after these peptides have been captured by specialized molecules in the host cell and displayed by them at the cell surface. The molecules that display peptide antigen to T cells are membrane glycoproteins encoded in a cluster of genes bearing the cumbersome name major histocompatibility complex, abbreviated to MHC.
The human MHC molecules were first discovered as the result of attempts to use skin grafts from donors to repair badly burned pilots and bomb victims during World War II. The patients rejected the grafts, which were recognized as being 'foreign.' It was soon appreciated from studies in mice that rejection was due to an immune response, and eventually genetic experiments using inbred strains of mice showed that rapid rejection of skin grafts is caused by differences in a single genetic region. Because they control the compatibility of tissue grafts, these genes became known as 'histocompatibility genes.' Later, it was found that several closely linked, and highly polymorphic genes specify histocompatibility, which led to the term major histocompatibility complex. The central role of the MHC in antigen recognition by T cells, was discovered later still, revealing the true physiological function of the proteins encoded by the MHC. This, in turn, led to an explanation for the major effect
on tissue compatibility for which they were named. We shall discuss these diverse functions of MHC molecules
Two major types of T cell recognize peptides bound to proteins of two different classes of MHC molecule.
There are two types of MHC molecule, called MHC class I and MHC class II. These differ in several subtle ways but share most of their major structural features. The most important of these is formed by the two outer extracellular domains of the molecule, which combine to create a long cleft in which a single peptide fragment is trapped during the synthesis and assembly of the MHC molecule inside the cell. The MHC molecule bearing its cargo of peptide is then transported to the cell surface, where it displays the peptide to T cells. The antigen receptors of T lymphocytes are specialized to recognize a foreign antigenic peptide fragment bound to an MHC molecule. A T cell with a receptor specific for the complex formed between that particular foreign peptide and MHC molecule can then recognize and respond to the antigen-presenting cell.
MHC molecules on the cell surface display peptide fragments of antigens.
MHC molecules are membrane proteins whose outer extracellular domains form a cleft in which a peptide fragment is bound. These fragments, which are derived from proteins degraded inside the cell, including foreign protein antigens, are bound by
the newly synthesized MHC molecule before it reaches the surface. There are two kinds of MHC molecule MHC class I and MHC class II with related but distinct structures and functions.
The most important differences between the two classes of MHC molecule lie not in their structure but in the source of the peptides that they trap and carry to the cell surface. MHC class I molecules collect peptides derived from proteins synthesized in the cytosol, and are thus able to display fragments of viral proteins on the cell surface. MHC class II molecules bind peptides derived from proteins in intracellular vesicles, and thus display peptides derived from pathogens living in macrophage vesicles or internalized by phagocytic cells and B cells
We shall see exactly how peptides from these different sources are made accessible to the two types of MHC molecule.
MHC class I molecules present antigen derived from proteins in the cytosol.
In cells infected with viruses, viral proteins are synthesized in the cytosol. Peptide fragments of viral proteins are transported into the endoplasmic reticulum (ER) where they are bound by MHC class I molecules, which then deliver the peptides to the
MHC class II molecules present antigen originating in intracellular vesicles.
Some bacteria infect cells and grow in intracellular vesicles. Peptides derived from such bacteria are bound by MHC class II molecules and transported to the cell surface (top row). MHC class II molecules also bind and transport peptides derived from antigen that has been bound and internalized by B-cell antigen receptor-mediated uptake into intracellular vesicles (bottom row).
Having reached the cell surface with their peptide cargo, the two classes of MHC molecule are recognized by different functional classes of T cell. MHC class I molecules bearing viral peptides are recognized by cytotoxic T cells, which then kill the infected cell; MHC class II molecules bearing peptides derived from pathogens
taken up into vesicles are recognized by TH1 or TH2 cells.
Cytotoxic T cells recognize antigen presented by MHC class I molecules and kill the cell.
The peptide:MHC class I complex on virus-infected cells is detected by antigen-specific cytotoxic T cells. Cytotoxic T cells are preprogrammed to kill the cells they recognize.
TH1 and helper T cells recognize antigen presented by MHC class II molecules.
On recognition of their specific antigen on infected macrophages, TH1 cells activate the macrophage, leading to the destruction of the intracellular bacteria (left panel). When helper T cells recognize antigen on B cells, they activate these cells to proliferate and differentiate into antibody-producing plasma cells (right panel).
The antigen-specific activation of these effector T cells is aided by co-receptors that distinguish between the two classes of MHC molecule; cytotoxic cells express the CD8 co-receptor that binds MHC class I molecules, whereas TH1 and TH2 cells express the CD4 co-receptor with specificity for MHC class II molecules. However, even before T
cells have encountered the specific foreign antigen that activates them to differentiate into effector cells, they express the appropriate co-receptor to match their receptor specificity. The maturation of T cells into either CD8 or CD4 T cells reflects the testing of T-cell receptor specificity that occurs during development, and the selection of T cells that can receive survival signals from self MHC molecules. Exactly how this selective process works, and how it maximizes the usefulness of the T cell repertoire is a central question in immunology.On recognizing their targets, the three types of T cell are stimulated to release different sets of effector molecules.
These can directly affect their target cells or help to recruit other effector cells in ways we shall discuss. These effector molecules include many cytokines, which have a crucial role in the clonal expansion of lymphocytes as well as in innate immune responses and in the effector actions of most immune cells; thus, understanding the
actions of cytokines is central to understanding the various behaviors of the immune system.
Defects in the immune system result in increased susceptibility to infection.
We tend to take for granted the ability of our immune systems to free our bodies of infection and prevent its recurrence. In some people, however, parts of the immune system fail. In the most severe of these immunodeficiency diseases, adaptive immunity is completely absent, and death occurs in infancy from overwhelming infection unless heroic measures are taken. Other less catastrophic failures lead to recurrent infections with particular types of pathogen, depending on the particular deficiency. Much has been learned about the functions of the different components of the human immune system through the study of these immunodeficiencies.
More than twenty-five years ago, a devastating form of immunodeficiency appeared, the acquired immune deficiency syndrome, or AIDS, which is itself caused by an infectious agent. This disease destroys the T cells that activate macrophages, leading to infections caused by intracellular bacteria and other pathogens normally controlled by these T cells. Such infections are the major cause of death from this increasingly prevalent immunodeficiency disease, which is discussed fully together with inherited immunodeficiencies. AIDS is caused by a virus, the human immunodeficiency virus, or HIV, that has evolved several strategies by which it not only evades but also subverts the protective mechanisms of the adaptive immune response. Such strategies are typical of many successful pathogens and we shall examine a variety of them. The conquest of many of the world's leading diseases, including malaria and diarrheal diseases (the leading killers of children) as well as the more recent threat from AIDS, depends upon a better understanding of the pathogens that cause them and their
interactions with the cells of the immune system.
Understanding adaptive immune responses is important for the control of allergies, autoimmune disease, and organ graft rejection.
Many medically important diseases are associated with a normal immune response directed against an inappropriate antigen, often in the absence of infectious disease. Immune responses directed at noninfectious antigens occur in allergy, where the antigen is an innocuous foreign substance, in autoimmune disease, where the response is to a self antigen, and in graft rejection, where the antigen is borne by a transplanted foreign cell. What we call a successful immune response or a failure, and whether the response is considered harmful or beneficial to the host, depends not
on the response itself but rather on the nature of the antigen.
Immune responses can be beneficial or harmful depending on the nature of the antigen.
Beneficial responses are shown in white, harmful responses in shaded boxes. Where the response is beneficial, its absence is harmful.
Allergic diseases, which include asthma, are an increasingly common cause of disability in the developed world, and many other important diseases are now recognized as autoimmune. An autoimmune response directed against pancreatic в cells is the leading cause of diabetes in the young. In allergies and autoimmune diseases, the powerful protective mechanisms of the adaptive immune response cause serious damage to the patient.
Immune responses to harmless antigens, to body tissues, or to organ grafts are, like all other immune responses, highly specific. At present, the usual way to treat these responses is with immunosuppressive drugs, which inhibit all immune responses, desirable or undesirable. If it were possible to suppress only those lymphocyte clones responsible for the unwanted response, the disease could be cured or the grafted organ protected without impeding protective immune responses. There is hope that this dream of antigenspecific immunoregulation to control unwanted immune
responses could become a reality, since antigen-specific suppression of immune responses can be induced experimentally, although the molecular basis of this suppression is not fully understood. We shall see how the mechanisms of immune regulation are beginning to emerge from a better understanding of the functional
subsets of lymphocytes and the cytokines that control them, and we shall discuss the present state of understanding of allergies, autoimmune disease, graft rejection, and immunosuppressive drugs
Vaccination is the most effective means of controlling infectious diseases.
Although the specific suppression of immune responses must await advances in basic research on immune regulation and its application, the deliberate stimulation of an immune response by immunization, or vaccination, has achieved many successes in the two centuries since Jenner's pioneering experiment.
Mass immunization programs have led to the virtual eradication of several diseases that used to be associated with significant morbidity (illness) and mortality (Fig. 1.33). Immunization is considered so safe and so important that most states in the United States require children to be immunized against up to seven common childhood diseases.
Impressive as these accomplishments are, there are still many diseases for which we lack effective vaccines. And even where vaccines for diseases such as measles or polio can be used effectively in developed countries, technical and economic problems can prevent their widespread use in developing countries, where mortality from these diseases is still high. The tools of modern immunology and molecular biology are being applied to develop new vaccines and improve old ones, and we shall discuss these advances. The prospect of controlling these important diseases is tremendously exciting. The guarantee of good health is a critical step toward population control and economic development. At a cost of pennies per person, great hardship and suffering can be alleviated.
Successful vaccination campaigns.
Diphtheria, polio, and measles and its consequences have been virtually eliminated in the United States, as shown in these three graphs. SSPE stands for subacute sclerosing panencephalitis, a brain disease that is a late consequence of measles infection in a few patients. When measles was prevented, SSPE disappeared 15 20 years later. However, as these diseases have not been eradicated worldwide,
immunization must be maintained in a very high percentage of the population to prevent their reappearance.
Lymphocytes have two distinct recognition systems specialized for detection of extracellular and intracellular pathogens. B cells have cell-surface immunoglobulin molecules as receptors for antigen and, upon activation, secrete the immunoglobulin as soluble antibody that provides defense against pathogens in the extracellular spaces of the body. T cells have receptors that recognize peptide fragments of intracellular pathogens transported to the cell surface by the glycoproteins of the major histocompatibility complex (MHC). Two classes of MHC molecule transport
peptides from different intracellular compartments to present them to distinct types of effector T cell: cytotoxic T cells that kill infected target cells, and TH1 cells and helper T cells that mainly activate macrophages and B cells, respectively. Thus, T cells are crucially important for both the humoral and cell-mediated responses of adaptive immunity. The adaptive immune response seems to have engrafted specific antigen recognition by highly diversified receptors onto innate defense systems, which have a central role in the effector actions of both B and T lymphocytes.
The vital role of adaptive immunity in fighting infection is illustrated by the immunodeficiency diseases and the problems caused by pathogens that succeed in evading or subverting an adaptive immune response. The antigenspecific
suppression of adaptive immune responses is the goal of treatment for important human diseases involving inappropriate activation of lymphocytes, whereas the specific stimulation of an adaptive immune response is the basis
of successful vaccination. The immune system defends the host against infection. Innate immunity serves as a first line of defense but lacks the ability to recognize certain pathogens and to provide the specific protective immunity that prevents reinfection. Adaptive immunity is based on clonal selection from a repertoire of lymphocytes bearing highly diverse antigenspecific
receptors that enable the immune system to recognize any foreign antigen. In the adaptive immune response, antigen-specific lymphocytes proliferate and differentiate into effector cells that eliminate pathogens. Host defense requires different recognition systems and a wide variety of effector mechanisms to seek out and destroy the wide variety of pathogens in their various habitats within the body and at its surface. Not only can the adaptive immune response eliminate a pathogen but, in the process, it also generates increased numbers of differentiated memory
lymphocytes through clonal selection, and this allows a more rapid and effective response upon reinfection. The regulation of immune responses, whether to suppress them when unwanted or to stimulate them in the prevention of infectious disease, is the major medical goal of research in immunology.
MODERN DIAGNOSTICS OF DRUG ALLERGY
Allergic reactions occur when an individual who has produced IgE antibody in response to an innocuous antigen, or allergen, subsequently encounters the same allergen. The allergen triggers the activation of IgE-binding mast cells in
the exposed tissue, leading to a series of responses that are characteristic of allergy. As we learned, there are circumstances in which IgE is involved in protective immunity, especially in response to parasitic worms, which are prevalent in less developed countries. In the industrialized countries, however, IgE responses to innocuous antigens predominate and allergy is an important cause of disease. Almost half the populations of North America and Europe have allergies to one or more common environmental antigens and, although rarely lifethreatening,
these cause much distress and lost time from school and work. Because of the medical importance of allergy in industrialized societies, much more is known about the pathophysiology of IgE-mediated responses than about the normal physiological role of IgE. The term allergy was originally defined by Clemens Von Pirquet as "an altered capacity of the body to react to a foreign substance," which was an extremely broad definition that included all immunological reactions. Allergy is now defined in a much more restricted manner as "disease following a response by the immune system to an otherwise innocuous antigen." Allergy is one of a class of immune system responses that are termed hypersensitivity reactions. These are harmful immune responses that produce tissue injury and may cause serious disease.
Hypersensitivity reactions were classified into four types by Coombs and Gell. Allergy is often equated with type I hypersensitivity (immediate-type hypersensitivity reactions mediated by IgE), and will be used in this sense here.
In this chapter we will first consider the mechanisms that favor the production of IgE. We then describe the pathophysiological consequences of the interaction between antigen and IgE that is bound by the high-affinity Fc receptor (Fc RI) on mast cells. Finally, we will consider the causes and consequences of other types of immunological hypersensitivity reactions.
IgE-mediated reactions to extrinsic antigens.
All IgE-mediated responses involve mast-cell degranulation, but the symptoms experienced by the patient can be very different depending on whether the allergen
is injected, inhaled, or eaten, and depending also on the dose of the allergen.
There are four types of hypersensitivity reaction mediated by immunological mechanisms that cause tissue damage.
Types I-III are antibody-mediated and are distinguished by the different types of antigens recognized and the different classes of antibody involved. Type I responses are mediated by IgE, which induces mastcell activation, whereas types II and III are mediated by IgG, which can engage Fc-receptor and complementmediated
effector mechanisms to varying degrees, depending on the subclass of IgG and the nature of the antigen involved. Type II responses are directed against cell-surface or matrix antigens, whereas type III responses are directed against soluble antigens, and the tissue damage involved is caused by responses triggered by immune
complexes. Type IV hypersensitivity reactions are T cell-mediated and can be subdivided into three groups. In the first group, tissue damage is caused by the activation of macro-phages by TH1 cells, which results in an inflammatory
response. In the second, damage is caused by the activation by TH2 cells of inflammatory responses in which eosinophils predominate; in the third, damage is caused directly by cytotoxic T cells (CTL).
The production of IgE.
IgE is produced by plasma cells located in lymph nodes draining the site of antigen entry or locally, at the sites of allergic reactions, by plasma cells derived from germinal centers developing within the inflamed tissue. IgE differs from other antibody isotypes in being located predominantly in tissues, where it is tightly bound to the mast-cell surface through the high-affinity IgE receptor known as Fc RI. Binding of antigen to IgE cross-links these receptors and this causes the release of chemical mediators from the mast cells, which may lead to the development of a type I hypersensitivity reaction. Basophils and activated eosinophils also express Fc RI; they can therefore displaysurface-bound IgE and also take part in the production of type I hypersensitivity reactions. The factors that lead to an antibody response dominated by IgE are still being worked out. Here we will describe our current understanding of these processes before turning to the question of how IgE mediates allergic reactions.
Allergens are often delivered transmucosally at low dose, a route that favors IgE production.
There are certain antigens and routes of antigen presentation to the immune system that favor the production of IgE. CD4 TH2 cells can switch the antibody isotype from IgM to IgE, or they can cause switching to IgG2 and IgG4 (human) or IgG1 and IgG3 (mouse). Antigens that selectively evoke TH2 cells that drive an IgE response are known as allergens.
Much human allergy is caused by a limited number of inhaled small-protein allergens that reproducibly elicit IgE production in susceptible individuals. We inhale many different proteins that do not induce IgE production; this raises the question of what is unusual about the proteins that are common allergens. Although we do not yet have a complete answer, some general principles have emerged. Most allergens are relatively small, highly soluble proteins that are carried on desiccated particles such as pollen grains or mite feces. On contact with the mucosa of the airways, for example, the soluble allergen elutes from the particle and diffuses into the mucosa.
Allergens are typically presented to the immune system at very low doses. It has been estimated that the maximum exposure of a person to the common pollen allergens in ragweed (Artemisia artemisiifolia) does not exceed 1 μg per year! Yet many people develop irritating and even life-threatening TH2-driven IgE antibody responses to these minute doses of allergen. It is important to note that only some of the people who are exposed to these substances make IgE antibodies against them.
Properties of inhaled allergens.
The typical characteristics of inhaled allergens are described in this
table. It seems likely that presenting an antigen transmucosally and at very low doses is a particularly efficient way of inducing TH2-driven IgE responses. IgE antibody production requires TH2 cells that produce interleukin-4 (IL-4) and IL-13 and it can be inhibited by TH1 cells that produce interferon-γ (IFN-γ). The presentation of low
doses of antigen can favor the activation of TH2 cells over TH1 cells, and many common allergens are delivered to the respiratory mucosa by inhalation of a low dose. The dominant antigen-presenting cells in the respiratory mucosa are myeloid dendritic cells. These take up and process protein antigens very efficiently and become activated in the process. This in turn induces their migration to regional lymph nodes and differentiation into professional antigen-presenting cells with co-stimulatory activity that favors the differentiation of TH2 cells.
Different cytokines induce switching to different isotypes.
The individual cytokines induce (violet) or inhibit (red) production of certain isotypes. Much of the inhibitory effect is probably the result of directed switching
to a different isotype. These data are drawn from experiments with mouse cells.
Enzymes are frequent triggers of allergy.
Several lines of evidence suggest that IgE is important in host defense against parasites. Many parasites invade their hosts by secreting proteolytic enzymes that break down connective tissue and allow the parasite access to host tissues, and it has been proposed that these enzymes are particularly active at promoting TH2 responses. This idea receives some support from the many examples of allergens that are enzymes. The major allergen in the feces of the house dust mite (Dermatophagoides pteronyssimus), which is responsible for allergy in approximately 20% of the North American population, is a cysteine protease homologous to papain, known as Der p 1. This enzyme has been found to cleave occludin, a protein component of intercellular tight junctions. This reveals one possible reason for the allergenicity of certain enzymes. By destroying the integrity of the tight junctions between epithelial cells, Der p 1 may gain abnormal access to subepithelial antigen-presenting cells, resident mast cells, and eosinophils.
The allergenicity of Der p 1 may also be promoted by its proteolytic action on certain receptor proteins on B cells and T cells. It has been shown to cleave the α subunit of the IL-2 receptor, CD25, from T cells. Loss of IL-2 receptor activity might interfere with the maintenance of TH1 cells, leading to a TH2 bias. The protease papain, derived from the papaya fruit, is used as a meat tenderizer and causes allergy in workers preparing the enzyme; such allergies are called occupational allergies. Another occupational allergy is the asthma caused by inhalation of the bacterial enzyme subtilisin, the 'biological' component of some laundry detergents.
Injection of enzymatically active papain (but not of inactivated papain) into mice stimulates an IgE response. A closely related enzyme, chymopapain, is used medically to destroy intervertebral discs in patients with sciatica; the
major (although rare) complication of this procedure is anaphylaxis, an acute systemic response to allergens.
Not all allergens are enzymes, however; for example, two allergens identified from filarial worms are enzyme inhibitors. Many protein allergens derived from plants have been identified and sequenced, but their functions are currently obscure. Thus, there seems to be no systematic association between enzymatic activity and allergenicity.
Scanning electron micrograph of D. pteronyssimus with some of its fecal pellets.
The enzymatic activity of some allergens enables penetration of epithelial barriers.
The epithelial barrier of the airways is formed by tight junctions between the epithelial cells. Fecal pellets from the house dust mite, D. pteronyssimus, contain a proteolytic enzyme, Der p 1, that acts as an allergen. It cleaves occludin, a protein that helps maintain the tight junctions, and thus destroys the barrier function of the epithelium. Fecal antigens can then pass through and be taken up by dendritic cells in subepithelial tissue. Der p 1 is taken up by dendritic cells, which are activated and move to lymph nodes (not shown), where they act as antigen-presenting cells, inducing the production of TH2 cells specific for Der p 1 and the production of Der p 1-specific IgE. Der p 1 may then bind directly to specific IgE on the resident mast cells, triggering mast-cell activation.
Class switching to IgE in B lymphocytes is favored by specific signals.
There are two main components of the immune response leading to IgE production. The first consists of the signals that favor the differentiation of naive TH0 cells to a TH2 phenotype. The second comprises the action of cytokines and co-stimulatory signals from TH2 cells that stimulate B cells to switch to producing IgE antibodies.
The fate of a naive CD4 T cell responding to a peptide presented by a dendritic cell is determined by the cytokines it is exposed to before and during this response, and by the intrinsic properties of the antigen, antigen dose, and route of presentation. Exposure to IL-4 favors the development of TH2 cells and to IL-12 favors that of TH1 cells. IgE antibodies are important in host defense against parasitic infections and this defense system is distributed anatomically mainly at the sites of entry of parasites under the skin, under the epithelial surfaces of the airways (the
mucosal-associated lymphoid tissues), and in the submucosa of the gut (the gut-associated lymphoid tissues). Cells of the innate and adaptive immune systems at these sites are specialized to secrete predominantly cytokines that drive
TH2 responses. The dendritic cells at these sites are of the myeloid phenotype; after taking up antigen they migrate to regional lymph nodes where their interaction with naive CD4 T cells drives the T cells to become TH2 cells, which secrete IL-4 and IL-10. It is not known how myeloid dendritic cells induce this differentiation. One possibility is that they express a particular set of cytokines and co-stimulatory molecules yet to be characterized. Another is that they activate a specialized subset of CD4 T cells, the NK1.1+ subset, that produce
abundant IL-4 that can induce CD4 T cells to differentiate into TH2 cells following stimulation by antigen. These in turn induce B cells to produce IgE.
Class switching of B cells to IgE production is induced by two separate signals, both of which can be provided by TH2 cells. The first of these signals is provided by the cytokines IL-4 or IL-13, interacting with receptors on the B-cell surface. These transduce their signal by activation of the Janus family tyrosine kinases JAK1
and JAK3 which ultimately lead to phosphorylation of the transcriptional regulator STAT6. Mice lacking functional IL-4, IL-13, or STAT6 all show impaired TH2 responses and IgE switching, demonstrating the key importance of these signaling pathways. The second signal for IgE class switching is a co-stimulatory interaction
between CD40 ligand on the T-cell surface with CD40 on the B-cell surface. This interaction is essential for all antibody class switching; patients with the X-linked hyper IgM syndrome have a deficiency of CD40 ligand and produce no IgG, IgA, or IgE.
The IgE response, once initiated, can be further amplified by basophils, mast cells, and eosinophils, which can also drive IgE production. All three cell types express Fc RI, although eosinophils only express it when activated. When these specialized granulocytes are activated by antigen cross-linking of their Fc RI-bound IgE, they
can express cell-surface CD40L and secrete IL-4; like TH2 cells, therefore, they can drive class switching and IgE production by B cells. The interaction between these specialized granulocytes and B cells can occur at the site of the allergic reaction, as B cells are observed to form germinal centers at inflammatory foci. Blocking this
amplification process is a goal of therapy, as allergic reactions can otherwise become self sustaining.
IgE class switching in B cells is initiated by TH2 cells, which develop in the presence of an early
burst of IL-4.
In mice, IL-4 is secreted early in some immune responses by a small subset of CD4 T cells (NK1.1+CD4 T cells) that interact with antigen-presenting cells bearing the nonclassical MHC class I-like molecule CD1 (first panel). Naive T cells being primed by their first encounter with antigen carry receptors for IL-4 (IL-4R) and are
driven to differentiate into TH2 cells in the presence of this early burst of IL-4 (second panel). When these TH2 effector cells interact with B cells specific for the same antigen, they induce isotype-switching so that IgE is produced.
Antigen binding to IgE on mast cells leads to amplification of IgE production.
IgE secreted by plasma cells binds to the high-affinity IgE receptor on mast cells (illustrated here), basophils, and activated eosinophils. When the surface-bound IgE is cross-linked by antigen, these cells express CD40L and secrete IL-4, which in turn binds to IL-4 receptors (IL-4R) on the activated B cell, stimulating isotype switching by B cells and the production of more IgE. These interactions can occur in vivo at the site of allergen-triggered inflammation, for example in bronchial-associated lymphoid tissue.
Genetic factors contribute to the development of IgE-mediated allergy, but environmental factors may also be important.
As many as 40% of people in Western populations show an exaggerated tendency to mount IgE responses to a wide variety of common environmental allergens. This state is called atopy and seems to be influenced by several genetic loci. Atopic individuals have higher total levels of IgE in the circulation and higher levels of eosinophils than their normal counterparts. They are more susceptible to allergic diseases such as hay fever and asthma. Studies of atopic families have identified regions on chromosomes 11q and 5q that appear to be important in determining atopy; candidate genes that could affect IgE responses are present in these regions. The candidate gene on chromosome 11 encodes the β subunit of the high-affinity IgE receptor, whereas on chromosome 5 there is a cluster of tightly linked genes that includes those for IL-3, IL-4, IL-5, IL-9, IL-12, IL-13, and granulocyte-macrophage colony-stimulating
factor (GM-CSF). These cytokines are important in IgE isotype switching, eosinophil survival, and mast-cell proliferation. Of particular note, an inherited genetic variation in the promoter region of the IL-4 gene is associated with raised IgE levels in atopic individuals; the variant promoter will direct increased expression of a reporter gene in
experimental systems. Atopy has also been associated with a gain-of-function mutation of the α subunit of the IL-4 receptor, which is associated with increased signaling following ligation of the receptor. It is too early to know how
important these different polymorphisms are in the complex genetics of atopy.
A second type of inherited variation in IgE responses is linked to the MHC class II region and affects responses to specific allergens. Many studies have shown that IgE production in response to particular allergens is associated with certain HLA class II alleles, implying that particular MHC:peptide combinations might favor a strong TH2 response. For example, IgE responses to several ragweed pollen allergens are associated with haplotypes containing the MHC class II allele DRB1*1501. Many individuals are therefore generally predisposed to make TH2 responses and
specifically predisposed to respond to some allergens more than others. However, allergies to common drugs such as penicillin show no association with MHC class II or the presence or absence of atopy. There is evidence that a state of atopy, and the associated susceptibility to asthma, rhinitis, and eczema, can be determined by different genes in different populations. Genetic associations found in one group of patients have frequently not been confirmed in patients of different ethnic origins. There are also likely to be genes that affect only particular aspects of allergic disease. For example, in asthma there is evidence for different genes affecting at least
three aspects of the disease phenotype IgE production, the inflammatory response, and clinical responses to particular types of treatment. Some of the best-characterized genetic polymorphisms of candidate genes associated with asthma are shown in, together with possible ways in which the genetic variation may affect the particular type of disease that develops and its response to drugs.
The prevalence of atopic allergy, and of asthma in particular, is increasing in economically advanced regions of the world, an observation that is best explained by environmental factors. The four main candidate environmental factors are changes in exposure to infectious diseases in early childhood, environmental pollution, allergen levels, and dietary changes. Alterations in exposure to microbial pathogens is the most plausible explanation at present for the increase in atopic allergy. Atopy is negatively associated with a history of infection with measles or hepatitis A virus,
and with positive tuberculin skin tests (suggesting prior exposure and immune response to Mycobacterium tuberculosis). In contrast, there is evidence that children who have had attacks of bronchiolitis associated with respiratory syncytial virus (RSV) infection are more prone to the later development of asthma. Children hospitalized with this disease have a skewed ratio of cytokine production away from IFN-γ towards IL-4, the cytokine that induces TH2 responses. It is possible that infection by an organism that evokes a TH1 immune response early in life might
reduce the likelihood of TH2 responses later in life and vice versa. It might be expected that exposure to environmental pollution would worsen the expression of atopy and asthma. The best evidence shows the opposite effect, however. Children from the city of Halle in the former East Germany, which has severe air pollution, had a lower prevalence of atopy and asthma than an ethnically matched population from Munich, exposed to much cleaner air. This does not mean that polluted air is not bad for the lungs. The children from Halle had a higher overall prevalence of respiratory disease than their counterparts from Munich, but this was predominantly not allergic in origin.
While it is clear that allergy is related to allergen exposure, there is no evidence that the rising prevalence of allergy is due to any systematic change in allergen exposure. Nor is there any evidence that changes in diet can explain the increase in allergy in economically advanced populations.
Candidate susceptibility genes for asthma.
May also affect response to bronchodilator therapy with β2-adrenergic agonists. Patients with alleles associated with reduced enzyme production failed to show a beneficial response to a drug inhibitor of 5-lipoxygenase. This is an example of a pharmacogenetic effect, in which genetic variation affects the response to medication.
Allergic reactions are the result of the production of specific IgE antibody to common, innocuous antigens. Allergens are small antigens that commonly provoke an IgE antibody response. Such antigens normally enter the body at very low doses by diffusion across mucosal surfaces and therefore trigger a TH2 response. The differentiation of naïve allergen-specific T cells into TH2 cells is also favored by the presence of an early burst of IL-4, which seems to be derived from a specialized subset of T cells. Allergen-specific TH2 cells produce IL-4 and IL-13, which drive
allergen-specific B cells to produce IgE. The specific IgE produced in response to the allergen binds to the highaffinity receptor for IgE on mast cells, basophils, and activated eosinophils. IgE production can be amplified by these cells because, upon activation, they produce IL-4 and CD40 ligand. The tendency to IgE over-production is influenced by genetic and environmental factors. Once IgE is produced in response to an allergen, reexposure to the allergen triggers an allergic response
Effector mechanisms in allergic reactions.
Allergic reactions are triggered when allergens cross-link preformed IgE bound to the high-affinity receptor Fc RI on mast cells. Mast cells line the body surfaces and serve to alert the immune system to local infection. Once activated, they induce inflammatory reactions by secreting chemical mediators stored in preformed granules, and by synthesizing leukotrienes and cytokines after activation occurs. In allergy, they provoke very unpleasant reactions to innocuous antigens that are not associated with invading pathogens that need to be expelled. The consequences of
IgE-mediated mast-cell activation depend on the dose of antigen and its route of entry; symptoms range from the irritating sniffles of hay fever when pollen is inhaled, to the life-threatening circulatory collapse that occurs in systemic anaphylaxis. The immediate allergic reaction caused by mast-cell degranulation is followed by a
more sustained inflammation, known as the late-phase response. This late response involves the recruitment of other effector cells, notably TH2 lymphocytes, eosinophils, and basophils, which contribute significantly to the immunopathology of an allergic response.
Mast-cell activation has different effects on different tissues.
Most IgE is cell-bound and engages effector mechanisms of the immune system by different pathways from other antibody isotypes.
Most antibodies are found in body fluids and engage effector cells, through receptors specific for the Fc constant regions, only after binding specific antigen through the antibody variable regions. IgE, however, is an exception as it is captured by the high-affinity Fc receptor in the absence of bound antigen. This means that IgE is mostly found fixed in the tissues on mast cells that bear this receptor, as well as on circulating basophils and activated eosinophils. The ligation of cell-bound IgE antibody by specific antigen triggers activation of these cells at the site of antigen
entry into the tissues. The release of inflammatory lipid mediators, cytokines, and chemokines at sites of IgEtriggered reactions results in the recruitment of eosinophils and basophils to augment the type I response. There are two types of IgE-binding Fc receptor. The first, Fc RI, is a high-affinity receptor of the immunoglobulin
superfamily that binds IgE on mast cells, basophils, and activated eosinophils. When the cellbound IgE antibody is cross-linked by a specific antigen, Fc RI transduces an activating signal. High levels of IgE, such as those that exist in subjects with allergic diseases or parasite infections, can result in a marked increase in Fc RI on the surface of mast cells, enhanced sensitivity of such cells to activation by low concentrations of specific antigen, and markedly increased IgE-dependent release of chemical mediators and cytokines.
The second IgE receptor, Fc RII, usually known as CD23, is a C-type lectin and is structurally unrelated to Fc RI; it binds IgE with low affinity. CD23 is present on many different cell types, including B cells, activated T cells, monocytes, eosinophils, platelets, follicular dendritic cells, and some thymic epithelial cells. This receptor was thought to be crucial for the regulation of IgE antibody levels; however, knockout mouse strains lacking the CD23 gene show no major abnormality in the development of polyclonal IgE responses. However the CD23 knockout mice
have demonstrated a role for CD23 in enhancing the antibody response to a specific antigen in the presence of that same antigen complexed with IgE. This antigen-specific, IgE-mediated enhancement of antibody responses fails to
occur in mice lacking the CD23 gene. This demonstrates a role for CD23 on antigen-presenting cells in the capture of antigen by specific IgE.
Mast cells reside in tissues and orchestrate allergic reactions.
Mast cells were described by Ehrlich in the mesentery of rabbits and named Mastzellen ('fattened cells'). Like basophils, mast cells contain granules rich in acidic proteoglycans that take up basic dyes. However, in spite of this resemblance, and the similar range of mediators stored in these basophilic granules, mast cells are derived from a different myeloid lineage than basophils and eosinophils. Mast cells are highly specialized cells, and are prominent residents of mucosal and epithelial tissues in the vicinity of small blood vessels and postcapillary venules, where they are well placed to guard against invading pathogens. Mast cells are also found in subendothelial connective tissue. They home to tissues as agranular cells; their final differentiation, accompanied by granule formation, occurs after they have arrived in the tissues. The major growth factor for mast cells is stem-cell factor (SCF), which acts on the cell-surface receptor c-Kit. Mice with defective c-Kit lack
differentiated mast cells and cannot make IgE-mediated inflammatory responses. This shows that such responses depend almost exclusively on mast cells.
Mast cells express Fc RI constitutively on their surface and are activated when antigens cross-link IgE bound to these receptors. Degranulation occurs within seconds, releasing a variety of preformed inflammatory mediators. Among these are histamine a short-lived vasoactive amine that causes an immediate increase in local blood flow and vessel permeability and enzymes such as mast-cell chymase, tryptase, and serine esterases. These enzymes can in turn activate matrix metalloproteinases, which break down tissue matrix proteins, causing tissue destruction. Large amounts of tumor necrosis factor (TNF)-α are also released by mast cells after activation. Some comes from stores in mast-cell granules; some is newly synthesized by the activated mast cells themselves. TNF-α activates endothelial cells, causing increased expression of adhesion molecules, which promotes the influx of inflammatory leukocytes and lymphocytes into tissues. On activation, mast cells synthesize and release chemokines, lipid mediators such as leukotrienes and plateletactivating
factor (PAF), and additional cytokines such as IL-4 and IL-13 which perpetuate the TH2 response. These mediators contribute to both the acute and the chronic inflammatory responses. The lipid mediators, in particular, act rapidly to cause smooth muscle contraction, increased vascular permeability, and mucus secretion, and also induce the influx and activation of leukocytes, which contribute to the late-phase response. The lipid mediators derive from membrane phospholipids, which are cleaved to release the precursor molecule arachidonic acid. This molecule can be
modified by two pathways to give rise to prostaglandins, thromboxanes, and leukotrienes. The leukotrienes, especially C4, D4, and E4, are important in sustaining inflammatory responses in the tissues. Many anti-inflammatory drugs are inhibitors of arachidonic acid metabolism. Aspirin, for example, is an inhibitor of the enzyme
cyclooxygenase and blocks the production of prostaglandins. IgE-mediated activation of mast cells thus orchestrates an important inflammatory cascade that is amplified by the recruitment of eosinophils, basophils, and TH2 lymphocytes. The physiological importance of this reaction is as a defense mechanism against certain types of infection. In allergy, however, the acute and chronic inflammatory reactions triggered by mast-cell activation have important pathophysiological consequences, as seen in
the diseases associated with allergic responses to environmental antigens.
IgE antibody cross-linking on mast-cell surfaces leads to a rapid release of inflammatory
Mast cells are large cells found in connective tissue that can be distinguished by secretory granules containing many inflammatory mediators. They bind stably to monomeric IgE antibodies through the very highaffinity Fc receptor I. Antigen cross-linking of the bound IgE antibody molecules triggers rapid degranulation,
releasing inflammatory mediators into the surrounding tissue. These mediators trigger local inflammation, which recruits cells and proteins required for host defense to sites of infection. These cells are also triggered during allergic reactions when allergens bind to IgE on mast cells.
Molecules released by mast cells on activation.
Mast cells produce a wide variety of biologically active proteins and other chemical mediators. The enzymes and toxic mediators listed in the first two rows are
released from the preformed granules. The cytokines, chemokines, and lipid mediators are synthesized after activation.
Eosinophils are normally under tight control to prevent inappropriate toxic responses.
Eosinophils are granulocytic leukocytes that originate in bone marrow. They are so called because their granules, which contain arginine-rich basic proteins, are colored bright orange by the acidic stain eosin. Only very small numbers of these cells are normally present in the circulation; most eosinophils are found in tissues, especially
in the connective tissue immediately underneath respiratory, gut, and urogenital epithelium, implying a likely role for
these cells in defense against invading organisms. Eosinophils have two kinds of effector function. First, on activation they release highly toxic granule proteins and free radicals, which can kill microorganisms and parasites but can also cause significant tissue damage in allergic reactions. Second, activation induces the synthesis of chemical mediators such as prostaglandins, leukotrienes, and cytokines, which amplify the inflammatory response by activating epithelial cells, and recruiting and activating more eosinophils and leukocytes. The activation and degranulation of eosinophils is strictly regulated, as their inappropriate activation would be very
harmful to the host. The first level of control acts on the production of eosinophils by the bone marrow. Few eosinophils are produced in the absence of infection or other immune stimulation. But when TH2 cells are activated, cytokines such as IL-5 are released that increase the production of eosinophils in the bone marrow and their release into the circulation. However, transgenic animals overexpressing IL-5 have increased numbers of eosinophils (eosinophilia) in the circulation but not in their tissues, indicating that migration of eosinophils from the circulation into tissues is regulated separately, by a second set of controls. The key molecules in this case are CC chemokines Most of these cause chemotaxis of several types of leukocyte, but two are specific for eosinophils and have been named eotaxin 1 and eotaxin 2.
The eotaxin receptor on eosinophils, CCR3, is a member of the chemokine family of receptors. This receptor also binds the CC chemokines MCP-3, MCP-4, and RANTES, which also induce eosinophil chemotaxis. The eotaxins and these other CC chemokines also activate eosinophils. Identical or similar chemokines also stimulate mast cells and basophils. For example, eotaxin attracts basophils and causes their degranulation, and MCP-1, which binds to CCR2, similarly activates mast cells in both the presence or absence of antigen. MCP-1 can also promote the differentiation of naive TH0 cells to TH2 cells; TH2 cells also carry CCR3 and migrate toward
eotaxin. These findings show that families of chemokines, as well as cytokines, can coordinate certain kinds of immune response. A third set of controls regulates the state of eosinophil activation. In their nonactivated state, eosinophils do not express high-affinity IgE receptors and have a high threshold for release of their granule contents. After activation by cytokines and chemokines, this threshold drops, Fc RI is expressed, and the number of Fcγ receptors and complement receptors on the cell surface also increases. The eosinophil is now primed to carry out its effector
activity, for example degranulation in response to antigen that cross-links specific IgE bound to Fc RI on the eosinophil surface. The potential of eosinophils to cause tissue injury is illustrated by rare syndromes due to abnormally large numbers of
eosinophils in the blood (hypereosinophilia). These syndromes are sometimes seen in association with T-cell lymphomas, in which unregulated IL-5 secretion drives a marked increase in the numbers of circulating eosinophils. The clinical manifestations of hypereosinophilia are damage to the endocardium and to nerves, leading to heart failure and neuropathy, both thought to be caused by the toxic effects of eosinophil granule proteins.
Eosinophils can be detected easily in tissue sections by their bright refractile orange coloration.
In this light micrograph, a large number of eosinophils are seen infiltrating a tumor of Langherhans' cells known as Langerhans' cell histiocytosis. The tissue section is stained with hematoxylin and eosin; it is the eosin that imparts the characteristic orange color to the eosinophils.
Eosinophils secrete a range of highly toxic granule proteins and other inflammatory mediators.
Hypereosinophilia can cause injury to the endocardium.
The top panel shows a section of the endocardium from a patient with hyper-eosinophilic syndrome. There is an organized fibrous exudate and the underlying endocardium is thickened by fibrous tissue. Although there are large numbers of circulating eosinophils, these cells are not seen in the injured endocardium, which is thought to be damaged by granules released from circulating eosinophils. The panel at the bottom shows two partly degranulated eosinophils (center) surrounded by
erythrocytes in a peripheral blood film.
Allergic reactions can be divided into an immediate response and a late-phase response.
A whealand- flare allergic reaction develops within a minute or two of superficial injection of antigen into the epidermis and lasts for up to 30 minutes. The reaction to an intracutaneous injection of house dust mite antigen is shown in the upper left panel and is labeled 'HDM;' the area labeled 'saline' shows the absence of any response to a control injection of saline solution. A more widespread edematous response, as shown in the upper right panel, develops approximately 8 hours later and can persist for some hours. Similarly, the response to an inhaled antigen can be divided into early and late responses (bottom panel). An asthmatic response in the lungs with narrowing of the airways caused by the constriction of bronchial smooth muscle can be measured as a fall in the forced expired volume of air in one second (FEV1). The immediate response peaks within minutes after antigen inhalation and then subsides.
Approximately 8 hours after antigen challenge, there is a late-phase response that also results in a fall in the FEV1. The immediate response is caused by the direct effects on blood vessels and smooth muscle of rapidly metabolized mediators such as histamine released by mast cells. The late-phase response is caused by the effects of an influx of inflammatory leukocytes attracted by chemokines and other mediators released by mast cells during and after the immediate response.
Eosinophils and basophils cause inflammation and tissue damage in allergic reactions.
In a local allergic reaction, mast-cell degranulation and TH2 activation cause eosinophils to accumulate in large numbers and to become activated. Their continued presence is characteristic of chronic allergic inflammation and they are thought to be major contributors to tissue damage. Basophils are also present at the site of an inflammatory reaction. Basophils share a common stem-cell precursor with
eosinophils; growth factors for basophils are very similar to those for eosinophils and include IL-3, IL-5, and GMCSF. There is evidence for reciprocal control of the maturation of the stem-cell population into basophils or eosinophils. For example, transforming growth factor (TGF)-β in the presence of IL-3 suppresses eosinophil
differentiation and enhances that of basophils. Basophils are normally present in very low numbers in the circulation and seem to have a similar role to eosinophils in defense against pathogens. Like eosinophils, they are recruited to the sites of allergic reactions. Basophils express Fc RI on the cell surface and, on activation by cytokines or antigen, they release histamine and IL-4 from the basophilic granules after which they are named. Eosinophils, mast cells, and basophils can interact with each other. Eosinophil degranulation releases major basic protein, which in turn causes degranulation of mast cells and basophils. This effect is augmented by any of the
cytokines that affect eosinophil and basophil growth, differentiation, and activation, such as IL-3, IL-5, and GM-CSF.
An allergic reaction is divided into an immediate response and a late-phase response.
The inflammatory response after IgE-mediated mast-cell activation occurs as an immediate reaction, starting within seconds, and a late reaction, which takes up to 8 12 hours to develop. These reactions can be distinguished clinically The immediate reaction is due to the activity of histamine, prostaglandins, and other preformed or
rapidly synthesized mediators that cause a rapid increase in vascular permeability and the contraction of smooth muscle. The late-phase reaction is caused by the induced synthesis and release of mediators including leukotrienes, chemokines, and cytokines from the activated mast cells. These recruit other leukocytes, including eosinophils and TH2 lymphocytes, to the site of inflammation. Although the late-phase reaction is clinically less marked than the immediate response, it is associated with a second phase of smooth muscle contraction, sustained edema, and the development of one of the cardinal features of allergic asthma: airway hyperreactivity to nonspecific
bronchoconstrictor stimuli such as histamine and methacholine. The late-phase reaction is an important cause of much serious long-term illness, as for example in chronic asthma. This is because the late reaction induces the recruitment of inflammatory leukocytes, especially eosinophils and TH2 lymphocytes, to the site of the allergen-triggered mast-cell response. This late response can easily convert into a
chronic inflammatory response if antigen persists and stimulates allergen-specific TH2 cells, which in turn promote eosinophilia and further IgE production.
The clinical effects of allergic reactions vary according to the site of mast-cell activation.
When reexposure to allergen triggers an allergic reaction, the effects are focused on the site at which mast-cell degranulation occurs. In the immediate response, the preformed mediators released are short-lived, and their potent effects on blood vessels and smooth muscles are therefore confined to the vicinity of the activated mast cell. The more sustained effects of the late-phase response are also focused on the site of initial allergen-triggered activation, and the particular anatomy of this site may determine how readily the inflammation can be resolved. Thus, the
clinical syndrome produced by an allergic reaction depends critically on three variables: the amount of allergenspecific IgE present; the route by which the allergen is introduced; and the dose of allergen. If an allergen is introduced directly into the bloodstream or is rapidly absorbed from the gut, the connective tissue mast cells associated with all blood vessels can become activated. This activation causes a very dangerous syndrome called systemic anaphylaxis. Disseminated mast-cell activation has a variety of potentially fatal effects: the widespread increase in vascular permeability leads to a catastrophic loss of blood pressure; airways constrict, causing
difficulty in breathing; and swelling of the epiglottis can cause suffocation. This potentially fatal syndrome is called anaphylactic shock. It can occur if drugs are administered to people who have IgE specific for that drug, or after an insect bite in individuals allergic to insect venom. Some foods, for example peanuts or brazil nuts, can cause systemic anaphylaxis in susceptible individuals. This syndrome can be rapidly fatal but can usually be controlled by the immediate injection of epinephrine, which relaxes the smooth muscle and inhibits the cardiovascular effects of anaphylaxis.
The most frequent allergic reactions to drugs occur with penicillin and its relatives. In people with IgE antibodies against penicillin, administration of the drug by injection can cause anaphylaxis and even death. Great care should be taken to avoid giving a drug to patients with a past history of allergy to that drug or one that is closely related
structurally. Penicillin acts as a hapten; it is a small molecule with a highly reactive β-lactam ring that is crucial for its antibacterial activity. This ring reacts with amino groups on host proteins to form covalent conjugates. When penicillin is ingested or injected, it forms conjugates with self proteins, and the penicillin-modified self peptides can provoke a TH2 response in some individuals. These TH2 cells then activate penicillin-binding B cells to produce IgE antibody to the penicillin hapten. Thus, penicillin acts both as the B-cell antigen and, by modifying self peptides, as the T-cell antigen. When penicillin is injected intravenously into an allergic individual, the penicillinmodified proteins can cross-link IgE molecules on the mast cells and cause anaphylaxis.
The dose and route of allergen administration determine the type of IgE-mediated allergic reaction that results.
There are two main anatomical distributions of mast cells: those associated with vascularized connective tissues, called connective tissue mast cells, and those found in submucosal layers of the gut and respiratory tract, called mucosal mast cells. In an allergic individual, all of these are loaded with IgE directed against specific allergens. The overall response to an allergen then depends on which mast cells are activated. Allergen in the bloodstream activates connective tissue mast cells throughout the body, resulting in the systemic release of histamine and other mediators. Subcutaneous administration of allergen activates only local connective tissue mast cells, leading to a local inflammatory reaction. Inhaled allergen, penetrating across epithelia, activates mainly mucosal mast cells, causing smooth muscle contraction in the lower airways; this leads to bronchoconstriction and difficulty in expelling inhaled air. Mucosal mast-cell activation also increases the local secretion of mucus by epithelial cells and causes irritation. Similarly, ingested allergen penetrates across gut epithelia, causing vomiting due to intestinal smooth muscle contraction and diarrhea due to outflow of fluid across the gut epithelium. Food allergens can also be
disseminated in the bloodstream, causing urticaria (hives) when the food allergen reaches the skin.
Allergen inhalation is associated with the development of rhinitis and asthma.
Inhalation is the most common route of allergen entry. Many people have mild allergies to inhaled antigens, manifesting as sneezing and a runny nose. This is called allergic rhinitis, and results from the activation of mucosal mast cells beneath the nasal epithelium by allergens such as pollens that release their protein contents, which can then diffuse across the mucus membranes of the nasal passages. Allergic rhinitis is characterized by intense itching and sneezing, local edema leading to blocked nasal passages, a nasal discharge, which is typically rich in eosinophils, and
irritation of the nose as a result of histamine release. A similar reaction to airborne allergens deposited on the conjunctiva of the eye is called allergic conjunctivitis. Allergic rhinitis and conjunctivitis are commonly caused by environmental allergens that are only present during certain seasons of the year. For example, hay fever is caused by a variety of allergens, including certain grass and tree pollens. Autumnal symptoms may be caused by weed pollen, such as that of ragweed. These reactions are annoying but cause little lasting damage. A more serious syndrome is allergic asthma, which is triggered by allergen-induced activation of submucosal mast
cells in the lower airways. This leads within seconds to bronchial constriction and increased secretion of fluid and mucus, making breathing more difficult by trapping inhaled air in the lungs. Patients with allergic asthma often need treatment, and asthmatic attacks can be life-threatening. An important feature of asthma is chronic
inflammation of the airways, which is characterized by the continued presence of increased numbers of TH2 lymphocytes, eosinophils, neutrophils, and other leukocytes.
Although allergic asthma is initially driven by a response to a specific allergen, the subsequent chronic inflammation seems to be perpetuated even in the apparent absence of further exposure to allergen. The airways become characteristically hyperreactive and factors other than reexposure to antigen can trigger asthma attacks. For example, the airways of asthmatics characteristically show hyperresponsiveness to environmental chemical irritants such as cigarette smoke and sulfur dioxide; viral or, to a lesser extent, bacterial respiratory tract infections can exacerbate the disease by inducing a TH2-dominated local response.
The acute response in allergic asthma leads to TH2-mediated chronic inflammation of the airways.
In sensitized individuals, cross-linking of specific IgE on the surface of mast cells by an inhaled allergen triggers them to secrete inflammatory mediators, causing increased vascular permeability, contraction of bronchial smooth muscle, and increased mucus secretion. There is an influx of inflammatory cells, including eosinophils and TH2 cells, from the blood. Activated mast cells and TH2 cells secrete cytokines that augment eosinophil activation and degranulation, which causes further tissue injury and the entry of more inflammatory cells. The result is chronic inflammation, which can cause irreversible damage to the airways.
Morphological evidence of chronic inflammation in the airways of an asthmatic patient.
Panel a shows a section through a bronchus of a patient who died of asthma; there is almost total occlusion of the airway by a mucus plug. In panel b, a close-up view of the bronchial wall shows injury to the epithelium lining the bronchus, accompanied by a dense inflammatory infiltrate that includes eosinophils, neutrophils, and lymphocytes.
Skin allergy is manifest as urticaria or chronic eczema.
The same dichotomy between immediate and delayed responses is seen in cutaneous allergic responses. The skin forms an effective barrier to the entry of most allergens but it can be breached by local injection of small amounts of allergen, for example by a stinging insect. The entry of allergen into the epidermis or dermis causes a localized
allergic reaction. Local mast-cell activation in the skin leads immediately to a local increase in vascular permeability, which causes extravasation of fluid and swelling. Mast-cell activation also stimulates the release of chemicals from local nerve endings by a nerve axon reflex, causing the vasodilation of surrounding cutaneous blood vessels, which causes redness of the surrounding skin. The resulting skin lesion is called a wheal-and-flare reaction. About 8 hours later, a more widespread and sustained edematous response appears in some individuals as a consequence of the latephase response. A disseminated form of the wheal-and-flare reaction, known as urticaria or hives, sometimes appears when ingested allergens enter the bloodstream and reach the skin. Histamine released by mast cells activated by allergen in the skin causes large, itchy, red swellings of the skin. Allergists take advantage of the immediate response to test for allergy by injecting minute amounts of potential
allergens into the epidermal layer of the skin. Although the reaction after the administration of antigen by intraepidermal injection is usually very localized, there is a small risk of inducing systemic anaphylaxis. Another standard test for allergy is to measure levels of IgE antibody specific for a particular allergen in a sandwich ELISA Although acute urticaria is commonly caused by allergens, the causes of chronic urticaria, in which the urticarial rash can recur over long periods, are less well understood. In up to a third of cases, it seems likely that chronic urticaria is
an autoimmune disease caused by autoantibodies against the α chain of Fc RI. This is an example of a type II hypersensitivity reaction in which an autoantibody against a cellular receptor triggers cellular activation, in this case causing mast-cell degranulation with resulting urticaria. A more prolonged inflammatory response is sometimes seen in the skin, most often in atopic children. They develop a persistent skin rash called eczema or atopic dermatitis, due to a chronic inflammatory response similar to that seen in the bronchial walls of patients with asthma. The etiology of eczema is not well understood. TH2 cells and IgE are involved, and it usually clears in adolescence, unlike rhinitis and asthma, which can persist throughout life.
Allergy to foods causes symptoms limited to the gut and systemic reactions.
When an allergen is eaten, two types of allergic response are seen. Activation of mucosal mast cells associated with the gastrointestinal tract leads to transepithelial fluid loss and smooth muscle contraction, causing diarrhea and vomiting. For reasons that are not understood, connective tissue mast cells in the dermis and subcutaneous tissues can also be activated after ingestion of allergen, presumably by allergen that has been absorbed into the bloodstream, and this results in urticaria. Urticaria is a common reaction when penicillin is given orally to a patient who already has
penicillin-specific IgE antibodies. Ingestion of food allergens can also lead to the development of generalized anaphylaxis, accompanied by cardiovascular collapse and acute asthmatic symptoms. Certain foods, most importantly peanuts, tree nuts, and shellfish, are particularly associated with this type of life-threatening response.
Allergy can be treated by inhibiting either IgE production or the effector pathways activated by crosslinking of cell-surface IgE.
The approaches to the treatment and prevention of allergy are set out. Two treatments are commonly used in clinical practice one is desensitization and the other is blockade of the effector pathways. There are also several approaches still in the experimental stage. In desensitization the aim is to shift the antibody response away
from one dominated by IgE toward one dominated by IgG; the latter can bind to the allergen and thus prevent it from activating IgE-mediated effector pathways. Patients are injected with escalating doses of allergen, starting with tiny amounts. This injection schedule gradually diverts the IgE-dominated response, driven by TH2 cells, to one driven by TH1 cells, with the consequent downregulation of IgE production. Recent evidence shows that desensitization is also associated with a reduction in the numbers of late-phase inflammatory cells at the site of the allergic reaction. A potential complication of the desensitization approach is the risk of inducing IgE-mediated allergic responses. An alternative, and still experimental, approach to desensitization is vaccination with peptides derived from common
allergens. This procedure induces T-cell anergy, which is associated with multiple changes in the T-cell phenotype, including downregulation of cytokine production and reduced expression of the CD3:T-cell receptor complex. IgE-mediated responses are not induced by the peptides because IgE, in contrast to T cells, can only recognize the intact antigen. A major difficulty with this approach is that an individual's responses to peptides are restricted by their MHC class II alleles; therefore, patients with different MHC class II molecules respond to different allergen-derived peptides. As the human population is outbred and expresses a wide variety of MHC class II alleles, the number of peptides required to treat all allergic individuals might be very large. Another vaccination strategy that shows promise in experimental models of allergy is the use of oligodeoxynucleotides rich in unmethylated cytosine guanine dinucleotides (CpG) as adjuvants for desensitization regimes. These oligonucleotides mimic bacterial DNA sequences known as CpG motifs and strongly promote TH1 responses. The signaling pathways that enhance the IgE response in allergic disease are also potential targets for therapy. Inhibitors of IL-4, IL-5, and IL-13 would be predicted to reduce IgE responses, but redundancy between some of the activities of these cytokines might make this approach difficult to implement in practice. A second approach to manipulating the response is to give cytokines that promote TH1-type responses. IFN-γ, IFN-α, IL-10, IL-12, and TGF-β have each been shown to reduce IL-4-stimulated IgE synthesis in vitro, and IFN-γ and IFN-α have been
shown to reduce IgE synthesis in vivo. Another target for therapeutic intervention might be the high-affinity IgE receptor. An effective competitor for IgE at
this receptor could prevent the binding of IgE to the surfaces of mast cells, basophils, and eosinophils. Candidate competitors include humanized anti-IgE monoclonal antibodies, which bind to IgE and block its binding to the receptor, and modified IgE Fc constructs that bind to the receptor but lack variable regions and thus cannot bind
antigen. Yet another approach would be to block the recruitment of eosinophils to sites of allergic inflammation. The eotaxin receptor CCR3 is a potential target for this type of therapy. The production of eosinophils in bone marrow and their exit into the circulation might also be reduced by a blockade of IL-5 action. The mainstays of therapy at present, however, are drugs that treat the symptoms of allergic disease and limit the inflammatory response. Anaphylactic reactions are treated with epinephrine, which stimulates the reformation of endothelial tight junctions, promotes the relaxation of constricted bronchial smooth muscle, and also stimulates the
heart. Inhaled bronchodilators that act on β-adrenergic receptors to relax constricted muscle are also used to relieve acute asthma attacks. Antihistamines that block the histamine H1 receptor reduce the urticaria that follows histamine release from mast cells and eosinophils. Relevant H1 receptors include those on blood vessels that cause increased permeability of the vessel wall, and those on unmyelinated nerve fibers that are thought to mediate the itching sensation. In chronic allergic disease it is extremely important to treat and prevent the chronic inflammatory tissue injury. Topical or systemic corticosteroids are used to suppress the chronic inflammatory changes
seen in asthma, rhinitis, and eczema. However, what is really needed is a means of converting the T-cell response to the allergenic peptide antigen from predominantly TH2 to predominantly TH1.
Approaches to the treatment of allergy.
Possible methods of inhibiting allergic reactions are shown. Two approaches are in regular clinical use. The first is the injection of specific antigen in desensitization regimes, which are believed to divert the immune response to the allergen from a TH2 to a TH1 type, so that IgG is produced in place of IgE. The second clinically useful approach is the use of specific inhibitors to block the synthesis or effects of
inflammatory mediators produced by mast cells.
The allergic response to innocuous antigens reflects the pathophysiological aspects of a defensive immune response whose physiological role is to protect against helminthic parasites. It is triggered by antigen binding to IgE antibodies bound to the high-affinity IgE receptor Fc RI on mast cells. Mast cells are strategically distributed beneath the mucosal surfaces of the body and in connective tissue. Antigen cross-linking the IgE on their surface causes them to release large amounts of inflammatory mediators. The resulting inflammation can be divided into early events, characterized by short-lived mediators such as histamine, and later events that involve leukotrienes, cytokines, and chemokines, which recruit and activate eosinophils and basophils. The late phase of this response can evolve into chronic inflammation, characterized by the presence of effector T cells and eosinophils, which is most clearly seen in chronic allergic asthma.
Immunological responses involving IgG antibodies or specific T cells can also cause adverse hypersensitivity reactions. Although these effector arms of the immune response normally participate in protective immunity to infection, they occasionally react with noninfectious antigens to produce acute or chronic hypersensitivity reactions.
Innocuous antigens can cause type II hypersensitivity reactions in susceptible individuals by binding to the surfaces of circulating blood cells.
Antibody-mediated destruction of red blood cells (hemolytic anemia) or platelets (thrombocytopenia) is an uncommon side-effect associated with the intake of certain drugs such as the antibiotic penicillin, the anti-cardiac arrhythmia drug quinidine, or the antihypertensive agent methyldopa. These are examples of type II hypersensitivity reactions in which the drug binds to the cell surface and serves as a target for anti-drug IgG antibodies that cause destruction of the cell. The anti-drug antibodies are made in only a minority of individuals and it is not clear why these individuals make them. The cell-bound antibody triggers clearance of the cell from the circulation, predominantly by tissue macrophages in the spleen, which bear Fcγ receptors.
Systemic disease caused by immune complex formation can follow the administration of large quantities of poorly catabolized antigens.
Type III hypersensitivity reactions can arise with soluble antigens. The pathology is caused by the deposition of antigen:antibody aggregates or immune complexes at certain tissue sites. Immune complexes are generated in all antibody responses but their pathogenic potential is determined, in part, by their size and the amount, affinity, and isotype of the responding antibody. Larger aggregates fix complement and are readily cleared from the circulation by the mononuclear phagocytic system. The small complexes that form at antigen excess, however, tend to deposit in
blood vessel walls. There they can ligate Fc receptors on leukocytes, leading to leukocyte activation and tissue injury. A local type III hypersensitivity reaction can be triggered in the skin of sensitized individuals who possess IgG antibodies against the sensitizing antigen. When antigen is injected into the skin, circulating IgG antibody that has diffused into the tissues forms immune complexes locally. The immune complexes bind Fc receptors on mast cells and other leukocytes, which creates a local inflammatory response with increased vascular permeability. The
enhanced vascular permeability allows fluid and cells, especially polymorphonuclear leukocytes, to enter the site from the local vessels. This reaction is called an Arthus reaction. The immune complexes also activate complement, releasing C5a, which contributes to the inflammatory reaction by ligating C5a receptors on leukocytes
. This causes their activation and chemotactic attraction to the site of inflammation. The
Arthus reaction is absent in mice lacking the α or γ chain of the FcγRIII receptor (CD16) on mast cells, but remains largely unperturbed in complementdeficient mice, showing the primary importance of FcγRIII in triggering inflammatory responses via immune complexes. A systemic type III hypersensitivity reaction, known as serum sickness, can result from the injection of large quantities of a poorly catabolized foreign antigen. This illness was so named because it frequently followed the
administration of therapeutic horse antiserum. In the preantibiotic era, antiserum made by immunizing horses was often used to treat pneumococcal pneumonia; the specific anti-pneumococcal antibodies in the horse serum would help the patient to clear the infection. In much the same way, antivenin (serum from horses immunized with snake venoms) is still used today as a source of neutralizing antibodies to treat people suffering from the bites of poisonous snakes.
Serum sickness occurs 7-10 days after the injection of the horse serum, an interval that corresponds to the time required to mount a primary immune response that switches from IgM to IgG antibody against the foreign antigens in horse serum. The clinical features of serum sickness are chills, fever, rash, arthritis, and sometimes
glomerulonephritis. Urticaria is a prominent feature of the rash, implying a role for histamine derived from mast-cell degranulation. In this case the mast-cell degranulation is triggered by the ligation of cellsurface FcγRIII by IgGcontaining
immune complexes. The course of serum sickness is illustrated. The onset of disease coincides with the development of antibodies against the abundant soluble proteins in the foreign serum; these antibodies form immune complexes with their antigens throughout the body. These immune complexes fix complement and can bind to and activate leukocytes bearing Fc and complement receptors; these in turn cause widespread tissue injury. The formation of immune complexes causes clearance of the foreign antigen and so serum sickness is usually a self-limiting disease. Serum
sickness after a second dose of antigen follows the kinetics of a secondary antibody response and the onset of disease occurs typically within a day or two. Serum sickness is nowadays seen after the use of anti-lymphocyte globulin, employed as an immunosuppressive agent in transplant recipients, and also, rarely, after the administration of streptokinase, a bacterial enzyme that is used as a thrombolytic agent to treat patients with a myocardial infarction or heart attack. A similar type of immunopathological response is seen in two other situations in which antigen persists. The first is when an adaptive antibody response fails to clear an infectious agent, for example in subacute bacterial endocarditis or chronic viral hepatitis. In this situation, the multiplying bacteria or viruses are continuously generating new antigen
in the presence of a persistent antibody response that fails to eliminate the organism. Immune complex disease ensues, with injury to small blood vessels in many tissues and organs, including the skin, kidneys, and nerves. Immune complexes also form in autoimmune diseases such as systemic lupus erythematosus where, because the
antigen persists, the deposition of immune complexes continues, and serious disease can result. Some inhaled allergens provoke IgG rather than IgE antibody responses, perhaps because they are present at relatively high levels in inhaled air. When a person is reexposed to high doses of such inhaled antigens, immune complexes form in the alveolar wall of the lung. This leads to the accumulation of fluid, protein, and cells in the
alveolar wall, slowing blood-gas interchange and compromising lung function. This type of reaction occurs in certain occupations such as farming, where there is repeated exposure to hay dust or mold spores. The disease that results is therefore called farmer's lung. If exposure to antigen is sustained, the alveolar membranes can become permanently damaged.
The deposition of immune complexes in local tissues causes a local inflammatory response known
as an Arthus reaction (type III hypersensitivity reaction).
In individuals who have already made IgG antibody against an antigen, the same antigen injected into the skin forms immune complexes with IgG antibody that has
diffused out of the capillaries. Because the dose of antigen is low, the immune complexes are only formed close to the site of injection, where they activate mast cells bearing Fcγ receptors (FcγRIII). As a result of mast-cell activation,
inflammatory cells invade the site, and blood vessel permeability and blood flow are increased. Platelets also accumulate inside the vessel at the site, ultimately leading to vessel occlusion.
Serum sickness is a classic example of a transient immune complex-mediated syndrome.
An injection of a foreign protein or proteins leads to an antibody response. These antibodies form immune complexes with the circulating foreign proteins. The complexes are deposited in small vessels and activate complement and phagocytes, inducing fever and the symptoms of vasculitis, nephritis, and arthritis. All these effects are transient and resolve when the foreign protein is cleared.
Delayed-type hypersensitivity reactions are mediated by TH1 cells and CD8 cytotoxic T cells.
Unlike the immediate hypersensitivity reactions described so far, which are mediated by antibodies, delayed-type hypersensitivity or type IV hypersensitivity reactions are mediated by antigen-specific effector T cells. These function in essentially the same way as during a response to an infectious pathogen. The causes and consequences of some syndromes in which type IV hypersensitivity responses predominate are listed These responses can be transferred between experimental animals by purified T cells or cloned T-cell lines.
Type IV hypersensitivity responses.
These reactions are mediated by T cells and all take some time to develop. They can be grouped into three syndromes, according to the route by which antigen passes into the body. In delayed-type hypersensitivity the antigen is injected into the skin; in contact hypersensitivity it is absorbed into the skin; and in gluten-sensitive enteropathy it is absorbed by the gut. The prototypic delayed-type hypersensitivity reaction is an artifact of modern medicine the tuberculin test This is used to determine whether an individual has previously been infected with
Mycobacterium tuberculosis. Small amounts of tuberculin a complex mixture of peptides and carbohydrates derived from M. tuberculosis are injected intradermally. In individuals who have previously been exposed to the bacterium, either by infection with the pathogen or by immunization with BCG, an attenuated form of M.
tuberculosis, a local T cell-mediated inflammatory reaction evolves over 24-72 hours. The response is mediated by TH1 cells, which enter the site of antigen injection, recognize complexes of peptide:MHC class II molecules on antigen-presenting cells, and release inflammatory cytokines, such as IFN-γ and TNF-β. The cytokines stimulate the expression of adhesion molecules on endothelium and increase local blood vessel permeability, allowing plasma and accessory cells to enter the site; this causes a visible swelling. Each of these phases takes several hours and so the fully developed response appears only 24-48 hours after challenge. The cytokines produced by the activated TH1 cells and their actions are shown.
The stages of a delayed-type hypersensitivity reaction.
The first phase involves uptake, processing, and presentation of the antigen by local antigen-presenting cells. In the second phase, TH1 cells that were primed by a
previous exposure to the antigen migrate into the site of injection and become activated. Because these specific cells are rare, and because there is little inflammation to attract cells into the site, it can take several hours for a T cell of
the correct specificity to arrive. These cells release mediators that activate local endothelial cells, recruiting an inflammatory cell infiltrate dominated by macrophages and causing the accumulation of fluid and protein. At this
point, the lesion becomes apparent. Very similar reactions are observed in several cutaneous hypersensitivity responses. These can be elicited by either CD4 or CD8 T cells, depending on the pathway by which the antigen is processed. Typical antigens that cause cutaneous hypersensitivity responses are highly reactive small molecules that can easily penetrate intact skin, especially if they cause itching that leads to scratching. These chemicals then react with self proteins, creating protein-hapten complexes that can be processed to hapten-peptide complexes, which can bind to MHC molecules that are recognized by T cells as foreign antigens. There are two phases to a cutaneous hypersensitivity response sensitization and elicitation. During the sensitization phase, cutaneous Langerhans' cells take up and process antigen,
and migrate to regional lymph nodes, where they activate T cells, with the consequent production of memory T cells, which end up in the dermis. In the elicitation phase, further exposure to the sensitizing chemical leads to antigen presentation to memory T cells in the dermis, with release of T-cell cytokines such as IFN-γ and IL- 17. This stimulates the keratinocytes of the epidermis to release cytokines such as IL-1, IL-6, TNF-α and GM-CSF, and CXC chemokines including IL-8, interferon-inducible protein (IP)-9, IP-10, and MIG (monokine induced by IFN-γ). These cytokines and chemokines enhance the inflammatory response by inducing the migration of monocytes into the lesion and their maturation into macrophages, and by attracting more T cells.
The delayed-type (type IV) hypersensitivity response is directed by chemokines and cytokines released by TH1 cells stimulated by antigen.
Antigen in the local tissues is processed by antigen-presenting cells and presented on MHC class II molecules. Antigen-specific TH1 cells that recognize the antigen locally at the site of injection release chemokines and cytokines that recruit macrophages to the site of antigen deposition. Antigen presentation by the newly recruited macrophages then amplifies the response. T cells can also affect local blood
vessels through the release of TNF-α and TNF-β, and stimulate the production of macrophages through the release of IL-3 and GM-CSF. Finally, TH1 cells activate macrophages through the release of IFN-γ and TNF-α, and kill macrophages and other sensitive cells through the cell-surface expression of the Fas ligand.
Elicitation of a delayed-type hypersensitivity response to a contact-sensitizing agent.
The contactsensitizing agent is a small highly reactive molecule that can easily penetrate intact skin. It binds covalently as a hapten to a variety of endogenous proteins, which are taken up and processed by Langerhans' cells, the major antigenpresenting cells of skin. These present haptenated peptides to effector TH1 cells (which must have been previously primed in lymph nodes and then have traveled back to the skin). These then secrete cytokines such as IFN-γ that
stimulate keratinocytes to secrete further cytokines and chemokines. These in turn attract monocytes and induce their maturation into activated tissue macrophages, which contribute to the inflammatory lesions depicted.
Blistering skin lesions on hand of patient with poison ivy contact dermatitis.
Langerhans' cells can take up antigen in the skin and migrate to lymphoid organs where they present it to T cells.
Langerhans' cells can ingest antigen by several means, but have no co-stimulatory activity. In the presence of infection, they take up antigen locally in the skin and then migrate to the lymph nodes. There they differentiate into dendritic cells that can no longer ingest antigen but now have co-stimulatory activity.
The rash produced by contact with poison ivy is caused by a T-cell response to a chemical in the poisonivy leaf called pentadecacatechol. This compound is lipid-soluble and can therefore cross the cell membrane and modify intracellular proteins. These modified proteins generate modified peptides within the cytosol, which are
translocated into the endoplasmic reticulum and are delivered to the cell surface by MHC class I molecules. These are recognized by CD8 T cells, which can cause damage either by killing the eliciting cell or by secreting cytokines such as IFN-γ. The well-studied chemical picryl chloride produces a CD4 T-cell hypersensitivity reaction. It modifies extracellular self proteins, which are then processed by the exogenous pathway into modified self peptides that bind to self MHC class II molecules and are recognized by TH1 cells. When sensitized TH1 cells recognize these complexes they can produce extensive inflammation by activating macrophages. As the chemicals in these examples are delivered by contact with the skin, the rash that follows is called a contact hypersensitivity reaction.
Some insect proteins also elicit delayed-type hypersensitivity response. However, the early phases of the host reaction to an insect bite are often IgE-mediated or the result of the direct effects of insect venoms. Important delayed-type hypersensitivity responses to divalent cations such as nickel have also been observed. These divalent cations can alter the conformation or the peptide binding of MHC class II molecules, and thus provoke a T-cell response. Finally, although this section has focused on the role of T cells in inducing delayed-type hypersensitivity reactions, there is
evidence that antibody and complement may also play a part. Mice deficient in B cells, antibody, or complement show impaired contact hypersensitivity reactions. These requirements for B cells, antibody, and complement may reflect their role in the early steps of the elicitation of these reactions.
Hypersensitivity diseases reflect normal immune mechanisms directed against innocuous antigens. They can be mediated by IgG antibodies bound to modified cell surfaces, or by complexes of antibodies bound to poorly catabolized antigens, as occurs in serum sickness. Hypersensitivity reactions mediated by T cells can be activated by modified self proteins, or by injected proteins such as those in the mycobacterial extract tuberculin. These T cellmediated responses require the induced synthesis of effector molecules and develop more slowly, which is why they
are termed delayed-type hyper-sensitivity. In some people, immune responses to otherwise innocuous antigens produce allergic or hypersensitivity reactions
upon reexposure to the same antigen. Most allergies involve the production of IgE antibody to common environmental allergens. Some people are intrinsically prone to making IgE antibodies against many allergens, and such people are said to be atopic. IgE production is driven by antigen-specific TH2 cells, which are initially primed in
the presence of a burst of IL-4 released by specialized T cells early in the immune response. The IgE produced binds to the high-affinity IgE receptor Fc RI on mast cells, basophils, and activated eosinophils. The physiological role of this system is to provide front-line defense against parasite pathogens but, in economically developed societies in which parasitic infections are uncommon, it is almost always involved in allergic reactions. Eosinophils and specific effector T cells have an extremely important role in chronic allergic inflammation, which is the major cause of the
chronic morbidity of asthma. Antibodies of other isotypes and antigen-specific effector T cells contribute to hypersensitivity to other antigens.
THERAPEUTIC DRUG MONITORING
The aim of therapeutic research is to find out an effective medication against the disease without any dangerous toxic action. The delicate problem is that very often the dose of drug confines with toxicity. Primary care physicians who understand
the complexities of drug dosing may be able to provide their patients with more effective pharmacologic therapy. Standard or empirical methods of dosing are appropriate for most agents. The evolution of pharmacokinetics and the recent
development of simple and reliable analytical technology has led to pharmacokinetic dosing, a more sophisticated and exact method of dosing certain agents. When used properly, measurements of plasma drug levels in the clinical setting may provide valuable information. While these drug levels often do allow more objective monitoring and titration of therapy the information also has the potential to be valueless or even misleading 1.
CONCEPT OF THERAPEUTIC DRUG MONITORING (TDM)
TDM is based on the principle that for some drugs there is a close relationship between the plasma level of the drug and its clinical effect. If such a relationship does not exit TDM is of little value. Like any diagnostic test, the measurement of plasma level is justified only when the information provided is of potential therapeutic benefit. The clinical value of plasma level monitoring dependson how precisely the treatment outcome can be defined. When therapeutic outcome can be objectively and replicably quantified, such as during antithrombotic therapy with coumarin derivatives, little additional information is gained by plasma levels. On the other hand when a precise therapeutic and point is difficult to define, monitoring of drug levels may be of considerable therapeutic assistance.
TDM will be useful if the following criteria are met:
1) the drug in question has a narrow therapeutic range,
2) a direct relationship exists between the drug or drug metabolite levels in plasma and the pharmacological or toxic effects,
3) the therapeutic effect can not be readily assessed by the clinical observation,
4) large individual variability in steady state plasma concentration exits at any given dose and
5) appropriate analytic techniques are available to determine the drug and metabolite levels.
TDM is unnecessary when
1) Clinical outcome is unrelated either to dose or to plasma concentration
2) dosage need not be individualized
3) the pharmacological effects can be clinically quantified
4) when concentration effect relationship remains unestablished,
5) drugs with wide therapeutic range such as beta blockers and calcium channel blockers.
MAJOR INDICATIONS FOR TDM:
While there may be specific individual circumstances for TDM, most indications can be summarized as follows :
1. Low therapeutic index
2. Poorly defined clinical end point
3. Non compliance
4. Therapeutic failure
5. Drugs with saturable metabolism
6. Wide variation in the metabolism of drugs
7. Major organ failure
8. Prevention of adverse drug effects
TCM OF ESTABLISHED VALUES:
1. Cardio active drugs : amiodarone, digoxin,
2. Antibiotics : gentamycin, amikacin
3. Antidepressants : lithium and tricyclic
4. Antiepileptic drugs : Phenytoin,
Valproic acid and
5. Bronchodilators : theophylline
chemotherapy : methotrexate
7. Immunosuppressives : cyclosporine
The methods currently available for analyzing data obtained in drug disposition studies are enormous. The fundamental procedures necessary for the quantification of the drug in the body are: recovery from body fluids, tissues, and organs, separation from the biological components, identification of the species concerned and finally
quantification. The analytical methodology employed should ideally: 1) distinguish between compounds of similar structure – unchanged drug and metabolites. 2) detect small amounts 3) be simple enough to use as a routine assay and 4) be unaffected by other drugs administered simultaneously.
Spectrophotometry and Fluorimetry: Prior to advent of GLC and HPLC, drug samples were analyzed by spectrophotometric methods. Solvent extraction schemes coupled with a spectrophotometric finish can still provide a much derived simplicity in assay procedure when the level of sensitivity required is not too low. i.e. in the ug/
ml range. However the drawbacks are large volume of samples, complex extraction procedures and interference by other compounds.
Thin layer chromatography (TLC): TLC possess adequate resolutions for identifying many drugs but it suffers from inability to quantify these drugs accurately and time consuming technique with inadequate sensitivity. However it is a useful
techniques in toxicology laboratory.
HPLC and GLS: These methods are highly specific, precise and sensitive. Besides multiple analyses can be done. The drawbacks are i) extraction step required ii) slow, single serial analysis, iii) column degenerates with time and iv) complex analyses require considerable processing. Out of these two, HPLC technique is superior
because thermolabile compounds can also be analyzed.
Radio immuno assay (RIA): It is sensitive, reasonably precise but requires the use of radionucleides. Cross reactivity with other closely reacted drugs is a potential problem with this technique. Besides it is not possible to find out the optically active isomer. The hazards of using radioactive materi0al is a considerable limitation of
Enzyme Immuno assay: These techniques offer some advantages over RIA in that no radioactive tracer is required; there is no need to separate the bound from the unbound fractions. However the potential for cross reactivity still exits. Burgess et
al3 compared serum phenytoin concentration in patients with normal renal functions and in patients with end stage renal disease using EMIT and GLC and found that in patients with renal insufficiency and EMIT values were 90% higher than GLC
values, Digoxin RIA remains as one of the most precise and sensitive methods for quantitation of digoxin in patients serum4.
Fluorescence polarization Immunoassay (FPIA) : This assay procedure combines
competitive protein binding with fluorescence polarization to give direct measurement without the need for a separation procedure. The advantages of this method are accuracy, precision and short turn around time. Apple et al5 compared three methods viz., FPIA, EMIT and HPLC for measurement of total and free phenytoin levels in uremic patients and found interferences in EMIT assays were minimal and that FPIA and HPLC determinations are in agreement.
INTERPRETATION OF SERUM DRUG CONCENTRATION AND ADJUSTMENT OF DOSAGE:
Drug concentration determinations must always be interpreted in the context of the clinical data. Therapeutic ranges are available but should be used only as a guide. Many factors alter the effect of a drug concentration at the site of action, e.g., serum concentration of digoxin that is therapeutic for most patients may be excessive for
a patient with hypokalemia. Furthermore range of serum drug concentration require adjustment when other drugs with synergistic or antagonistic actions are administered concomitantly.
MAJOR CAUSES OF UNEXPECTED SERUM CONCENTRATION IN PATIENTS:
The most important causes of unexpected serum concentrations are non compliance,
inappropriate dosage, malabsorption, poor bioavailability, drug interactions, hepatic or renal disease altered protein binding and genetic factors. If these factors can not be eliminated, a dosage adjustment is required. For drugs with linear kinetics the following formulae may be used:
Desired drug concentration
New dose = Old dose X ________________________
Old drug concentration
Timing of Sample collection: The importance of proper timing of a sample is not given sufficient attention while ordering measurement of a plasma concentration1.
The best sampling time is in the predose or through phase just prior to a maintenance dose, when a drug is administered by multiple oral doses. This principle is important for digitalis which is administered on a once daily basis in the morning.
For drugs with a long half life such a phenytoin atleast 4 to 5 half lives must elapse before a sample is taken. A knowledge of usual half life ranges will thus be useful.
Sample timing for some important drugs:
a)Phenytoin: Since phenytoin has a long half life a single daily dose may be employed and so the timing of concentration monitoring is not critical 6.
b) Carbamazepine: Its half life may be as long as 48 h following a single dose. A through concentration taken just after a dose together with a peak level three hours later is ideal.
c) Digoxin: The measurement must be made atleast six hours after a dose to avoid inappropriate high levels.
d) Theophylline: This drug has a narrow therapeutic index and timing of sampling is not critical if the patient is receiving one of the slow release formulations.
e) Lithium: A 12 hr sample gives the most precise guide to dosage adjustment.
f) Phenobarbitone: Any time sample is sufficient
g) Gentamicin: Pre dose peak; 0.5 hr after i.v. and
1 hr after i.m. administration.
It is one of the factors to be considered while interpreting TDM data. It is important to know the duration of drug therapy, dosage and when the last dosage was taken.
Many drugs are biotransformed into compounds that are pharmacologically active.
When evaluating the therapeutic effect of such drugs, the relative contributions of all active substances present in the serum must be integrated e.g. imipramine is biotransformed to the active metabolite – desipramine.
EFFECT OF DISEASE STATES:
Acute or chronic disease alters drug clearance patterns. Thus drug concentrations may be elevated or depressed depending on the pathophysiology of the system involved, e.g., liver disease impairs the clearance of drugs dependent upon conversion to more water soluble compounds. Congestive cardiac failure can
precipitate elevated drug levels of agents dependent on hepatic metabolism for clearance.
FREE DRUG MONITORING:
Development of new filtration devices (equilibrium dialysis, ultrafiltration,
ultracentrifugation) has made it possible to measure free unbound drug levels in serum. The advantages are that the free concentrations is independent of changes in plasma binding and is the pharmacologically active concentration. The disadvantages are that it is time consuming, expensive and therapeutic ranges do not yet exist for many drugs.
USE OF SALIVA IN DRUG MONITORING:
The concentration of a drug in saliva is proportional to the concentration of the unbound rather than to the total of bound and unbound drugs in plasma . The practice of measuring drugs in saliva is appealing because it is non invasive.
However it has its limitations viz., some substances such as lithium are actively secreted into the saliva rather than by passive process. Drug binding to
salivary proteins may produce discrepancies in plasma/salivary ratios, e.g. phenytoin. Drugs may also bind to oral cell debris, e.g. propranolol, Salivary flow may be reduced in patients taking anti cholinergic drugs. Preparations used to stimulate salivary flow might interfere with drug estimation e.g. lemon flavored sweets interfere with amitryptyline estimations.
EFFECT OF AGE:
Variability in response to drugs occurs at extremes of age. Elderly patients are more
sensitive to the CNS depressant effect of drugs but are less sensitive to cardiovascular effects of propranolol. On the other hand young children are more sensitive to CNS depression effects of morphine. However more data are needed on the
effects of age on pharmacokinetic and pharmacodynamics of drugs to allow optional
individualization of dosage.
Little has ben published on the monitoring of plasma drug levels during pregnancy. Plasma drug levels of phenytoin and phenobarbitone tend to reduce during pregnancy.
The measurement of drug levels in body fluids must be cost effective. The cost of performing an individual test is determined by the summing equipment, personnel, supply and overhead expenditure for a given period of time and dividing
that amount but the number of assays performed in the same time interval. The fee charges is then determined by the test’s cost plus desired profit. The foregoing calculations produce an unreasonably expensive fee although high fee for
unique tests requiring special methods may not be unreasonable. Cost-benefit analysis of gentamicin dosage regimens of burn patients with gram negative septicaemia showed that a cost benefit ratio of 8.7 to 1 10 with decreased mortality and increased economic productivity. Mungall et al11 showed that use of clinical pharmacokinetics by therapeutic drug monitoring service offered substantial benefits like fewer adverse reactions, shorter intensive care unit stay and shorter overall
CLINICAL USEFULNESS OF TDM:
TDM data provides the clinician with greater insight into the factors determining the patients response to drug therapy. For example when a patient fails to respond to a usual therapeutic dose, measurement of plasma level can help to distinguish a noncompliant patient and a patient who is a true non-responder. TDM also provides
useful information regarding individual variations in drug utilization patterns and alteration in drug utilization as a consequence of altered
physiological state or disease process. TDM is a useful adjunct in treating many
patients provided the potential pit falls and problems are considered.
PREVENTABLE ADVERSE DRUG REACTIONS: A FOCUS ON DRUG INTERACTIONS
The first case we will consider is that of the potentially lethal arrhythmia, torsades de pointes (French for “twisting of the points”), occurring in a young woman and in association with the administration of the antihistamine terfenadine (Seldane).
This ECG is a classic example of torsades de pointes, and illustrates how the arrhythmia appears on the ECG. The ventricular complexes during this rhythm tend to show a series of “points going up” followed by “points going down,” often with a narrow waist between. Torsades de pointes is a form of ventricular tachycardia frequently caused by medications, but can occur in patients with an inherited disorder of cardiac ion channels, i.e., the congenital long QT syndrome. Clinically, torsades de pointes is a syndrome in which rapid polymorphic ventricular tachycardia (very often, but not always, showing the twisting of the points pattern) occurs in the setting of prolongation of cardiac repolarization (QT interval prolongation on the ECG).
Recognition and reporting of this arrhythmia in association with terfenadine (Seldane), astemizole (Hismanal), cisapride (Propulsid), grepafloxacin (Raxar), levomethadyl (Orlaam) and mibefradil (Posicor), ultimately led to these medications’ removal from the regular prescription market.
A 39-year-old female was evaluated for episodes of syncope and light-headedness that began two days prior to her hospital admission. The history was consistent with possible cardiovascular causes, and the patient was admitted and placed on telemetry where the preceding rhythm strip was observed. Ten days prior to admission, she had been prescribed terfenadine (Seldane - an antihistamine) 60 mg twice-a-day and cefaclor (Ceclor - a cephalosporin antibiotic) 250 mg three-times-a-day. On the eighth day of terfenadine therapy the patient began a self-medicated course of ketoconazole (Nizoral - an azole antifungal drug) at 200 mg twice-a-day for vaginal candidiasis. She was also taking medroxyprogesterone acetate at a dosage of 2.5 mg a-day. Upon admission to the hospital, the patient was noted to have a QTc (Bazett correction) interval of 655 milliseconds (normal is less than 440 milliseconds). During the hospitalization, the patient experienced near syncopal episodes associated with torsades de pointes observed on ECG telemetry.
After discontinuing the medications, the QTc interval normalized. She had no further episodes of torsades de pointes, and she was discharged with no recurrence of syncope.
This figure illustrates the time course of the medications that the patient took. Her symptoms started shortly after she began taking ketoconazole.