Substances that can give rise to wheal and flare responses in the skin and to the symptoms of allergic disease are derived from many different sources (see http://www.allergen.org/). When purified they are almost all found to be proteins and their sizes range from 10–40 kDa. These proteins are all freely soluble in aqueous solution, but have many different biological functions including digestive enzymes, carrier proteins, calycins, and pollen recognition proteins.

Any allergen can be described or classified by its source, route of exposure, and nature of the specific protein (Fig. 23.3).

Fig. 23.3 Properties of allergens

Patients who become ‘allergic’ to one of the well-recognized sources of allergens have actually produced an IgE antibody response to one or more of the proteins produced by mites, trees, grass, cats, or fungi. The proteins are predominantly water soluble with a molecular weight (MW) ranging from 10–40 kDa. In many cases the function of the proteins is known, but it is not clear whether function such as enzymic activity alters the ability of these proteins to induce an allergic response. The properties of the particles carrying these allergens are very important because they influence both how much becomes airborne, and also where the allergen is deposited in the respiratory tract. The dimensions of the particles airborne vary from ≤2 μm for Aspergillus or Penicillium spores to ≥20 μm for mite fecal pellets and some pollen grains. (Sizes are given as diameter in μm. For a full list of allergens see http://www.allergen.org/.)

Extracts used for skin testing or in-vitro measurement of IgE antibodies are made from the whole material, which contains multiple different proteins, any of which can be an allergen. Indeed, it is clear that individual patients can react selectively to one or more of the different proteins that are present in an extract.

Q. Name one genetic factor that determines whether an individual can make an immune response to a specific allergen

A. The individual’s MHC haplotypes determine which antigens and antigen fragments are presented to T cells.

Estimates of exposure can be made either by visual identification of particles (e.g. pollen grains or fungal spores) or by immunoassay of the major allergens (e.g. Fel d1 or Der p1).

IgE is distinct from the other dimeric immunoglobulins

In 1921, Küstner, who was allergic to fish, injected his own serum into the skin of Prausnitz, who was allergic to grass pollen but not fish, and demonstrated that it was possible to passively transfer immediate hypersensitivity (the Prausnitz–Küstner or P–K test). Prausnitz also noticed that an immediate wheal and flare occurred at the site of passive sensitization when he ate fish. This showed that some protein or part of fish proteins sufficient to trigger mast cells can be absorbed into the circulation.

Over the next 30 years it was established that P–K activity was a general property of the serum of patients with immediate hypersensitivity and that it was allergen specific (i.e. it behaved like an antibody).

In 1967 Ishizaka and his colleagues purified the P–K activity from a patient with ragweed hayfever and proved that this was a novel isotype of immunoglobulin – IgE. However, it was obvious that the concentration of this immunoglobulin isotype in serum was very low i.e. ≤1 μg/mL.

IgE is distinct from the other dimeric immunoglobulins because it has:

• an extra constant region domain;

• a different structure to the hinge region; and

• binding sites for both high- and low-affinity IgE receptors, FcεRI and FcεRII, respectively (see Fig. 3.19).

The primary cells that bear FcεRI are mast cells and basophils, which are the only cells in the human that contain significant amounts of histamine.

Low-affinity receptors for IgE – FcεRII or CD23 – are also present on B cells and may play a role in antigen presentation.

In addition in atopic dermatitis dendritic cells in skin can express a high-affinity receptor for IgE, but this receptor lacks the β chain of FcεRI.

The properties of IgE can be separated into three areas:

• the characteristics of the molecule including its half-life and binding to IgE receptors;

• the control of IgE and IgG antibody production by T cells; and

• the consequences of allergen cross-linking IgE on the surface of mast cells or basophils.

T cells control the response to inhalant allergens

IgE production is dependent on TH2 cells

Experiments in animals have established that the production of IgE is dependent on T cells. It is also clear that T cells can suppress IgE production.

T cells that suppress TH2 responses including IgE production:

• act predominantly by producing interferon-γ (IFNγ); and

• are produced when the animal (e.g. mouse, rat, or rabbit) is primed in the presence of Freund’s complete adjuvant.

This adjuvant, which includes bacterial cell walls and probably bacterial DNA, is a very potent activator of macrophages.

With the discovery of TH1 and TH2 cells, it became clear that IgE production is dependent on TH2 cells and that any priming that generates a TH1 response will inhibit IgE production.

The main cytokines that are specifically relevant to a TH1 response include:

• interleukin-12 (IL-12) produced by macrophages; and

• IFNγ produced by T cells.

By contrast, the primary cytokines relevant to a TH2 response are:

• IL-4 and IL13;

• IL-5; and

• IL-10 (Fig. 23.5).

Fig. 23.5 T cell differentiation during human immune responses

The differentiation of TH cells depends on the antigen source, the quantity of allergen, and the cytokines produced. Bacterial antigens or a high dose of antigen will induce IL-12 from macrophages. In addition, the developing TH1 cells produce IFNγ, which further enhances the production of TH1 cells. Low-dose antigen without adjuvant will induce TH2 cells, which produce both IL-4 and IL-5. IL-4 plays a role in (i) enhancing the growth of TH2 cells; (ii) the expression of the gene for IgE. In turn IgE binds to the high-affinity receptor for IgE (FcεRI) on mast cells. IL-5 plays a critical role in the production of eosinophils.

It is clear from experiments in mice and humans that the expression of the gene for IgE is dependent on IL-4. Thus, if immature human B cells are cultured with anti-CD40 and IL-4, they will produce IgE antibodies.

Cytokines regulate the production of IgE

In humans IgE antibodies are the dominant feature of the response to a select group of antigens and most other immune responses do not include IgE.

The classical allergens are inhaled in very small quantities (5–20 ng/day) either perennially indoors or over a period of weeks or months outdoors. Immunization of mice with repeated low-dose antigen is a very effective method of inducing IgE responses.

By contrast, the routine immunization of children with diphtheria and tetanus toxoid does not induce persistent production of IgE antibodies. This is clear because we do not routinely take precautions against anaphylaxis when administering a booster injection of tetanus.

Q. In which tissues do TH2-type immune responses predominate, and in which tissues are TH1 responses promoted?

A. TH2 responses predominate in mucosal tissues, whereas TH1 responses predominate in the skin and CNS (see Chapter 12).

As T cells differentiate, TH1 cells express the functional IL-12 receptor with the IL-12 β2 chain. By contrast, TH2 cells express only part of the IL-12 receptor and this part is non-functional.

IL-4 is important in the differentiation of TH2 cells and is also a growth factor for these cells. Because it is produced by TH2 cells, it is at least in part acting on the cell that produced it (i.e. in an autocrine fashion). The interaction of IL-4 with T cells can be blocked either with:

• an antibody to IL-4; or

• a soluble form of the IL-4 receptor (IL-4R).

The release of soluble IL-4R from T cells may be a natural mechanism for controlling T cell differentiation. However, recent evidence suggests that in-vivo responses are controlled by T cells producing either IL-10 or transforming growth factor-β (TGFβ).

Both IgE and IgG4 are dependent on IL-4

The genes for immunoglobulin heavy chains are in sequence on chromosome 14. The gene for ε occurs directly following the gene for γ4. Both of these isotypes are dependent on IL-4 and they may be expressed sequentially (Fig. 23.6).

Fig. 23.6 Immunoglobulin genes on chromosome 14: two models of rearrangement to allow expression of the IgE gene

Switch regions and heavy chain genes for immunoglobulin are arranged sequentially on chromosome 14. Both Cγ4 and Cε expression are dependent on IL-4 produced by T cells. The switch region of IgE often includes elements from Sγ4 indicating that the switching occurs sequentially. However, IgG4 responses can occur without IgE antibody responses.

The mechanisms by which IgG4 is controlled separately from IgE are not well understood, but this may include a role for IL-10. Thus, immunotherapy for patients with anaphylactic sensitivity to honey bee venom will induce IL-10 production by T cells, decreased IgE, and increased IgG4 antibodies to venom antigens.

Recently, it has been shown that children raised in a house with a cat can produce an IgG response, including IgG4 antibody, without becoming allergic. A modified TH2 response (increased IgG4 and decreased IgE) therefore represents an important mechanism of tolerance to allergens (Fig. 23.7). IgG4 antibody responses without IgE antibody are a feature of immunity/tolerance to insect venom, rat urinary allergens, and food antigens as well as cat allergens.

Fig. 23.7 Modified TH2 response

The TH2 response includes T effector cells as well as help for IgE and IgG4 antibody production. In turn IgE plays a major role in triggering mast cells. However, increasing evidence shows that higher doses of allergen (e.g. bee venom, cat dander, or rat urine) can induce a modified or tolerant TH2 response. This response includes IgG4 antibodies, but not IgE. The cytokine IL-10 may well play a role in enhancing IgG4 antibody while suppressing production of IgE.

Characteristics of allergens

Allergens have similar physical properties

In mice a wide range of proteins can be used to induce an IgE antibody response. The primary factors that influence the response are:

• the strain of mouse;

• the dose; and

• adjuvants used.

Thus, repeated low-dose immunization with alum or pertussis (but not complete Freund’s adjuvant) will produce IgE responses. However, the dose necessary to induce an optimal response varies greatly from one strain to another.

The allergens that have been defined have similar physical properties (i.e. freely soluble in aqueous solution with a molecular weight of 10–40 kDa), but are diverse biologically. Cloning has revealed sequence homology between allergens and proteins including calycins, pheromone binding proteins, enzymes, and pollen recognition proteins. Although many of the allergens have homology with known enzymes, this is not surprising because enzymic activity is an important property of proteins in general. Some important allergens, for example Der p2 from mites, Fel d1 from cats, and Amb a5 from ragweed pollen, have neither enzymic activity nor homology with known enzymes. Thus, enzymic activity is not essential for immunogenicity.

Nevertheless, the group I allergens of dust mites are cysteine proteases and in several model situations it has been shown that this enzymic activity influences the immunogenicity of the protein. Thus cleavage of CD23 or CD25 on lymphocytes by Der p1 can enhance immune responses. Alternatively, it has been shown that Der p1 can disrupt epithelial junctions and alter the entry of proteins through the epithelial layer. The interest in this property is increased because many different mite allergens are inhaled together in the fecal particles so the enzymic activity of one protein (i.e. Der p1) could facilitate either the physical entry or the immune response to other mite proteins.

The primary characterization of allergens relates to their route of exposure. The routes includes:

• inhaled allergens;

• foods;

• drugs;

• antigens from fungi growing on the body (e.g. Aspergillus spp.); and

• venoms.

The routes are important because they define the ways in which the antigens are presented to the immune system. Antigen presentation may well be the site at which genetic influences play the biggest role, the properties of the different groups of allergen need to be considered separately.

The inhalant allergens cause hayfever, chronic rhinitis, and asthma

The inhalant allergens are the primary causal agents in hayfever, chronic rhinitis, and asthma among school-aged children and young adults and they play an important role in atopic dermatitis.

Allergens can only become airborne in sufficient quantity to cause an immune response or symptoms when they are carried on particles. Pollen grains, mite fecal particles, particles of fungal hyphae or spores, and animal skin flakes (or dander) are the best defined forms in which allergens are inhaled (Fig. 23.8).

Fig. 23.8 Particles carrying airborne allergens – mite fecal pellets and pollen grains

The dust mite is the most important source of allergen in house dust, largely as fecal particles (1). A mite is shown in (2) with pollen grains lower left and fecal particles upper right. The mite is approximately 300 μm in length (i.e. just visible but not small enough to become airborne). Mite fecal particles are approximately 10–40 μm in diameter and become airborne during domestic disturbance. Pollen grains are similar in size to mite fecal particles (i.e. approximately 30 μm in diameter). The important allergic sources of pollen (i.e. grass, ragweed, and trees) are wind pollinated and the grains are designed to travel in the air for long distances.

In each case it is possible to define the approximate particle size and the quantity of protein on the particle as well as the speed with which the proteins in the particle dissolve in aqueous solution (see Fig. 23.3).

Thus, for grass pollen, mite fecal pellets, and cat dander:

• the relevant allergens are present in high concentrations within the particles (up to 10 mg/cm³);

• the particles are ‘large’ (i.e. 3–30 μm diameter); and

• the allergens elute rapidly in aqueous solution.

The allergens within these particles will be delivered to the nasal epithelium and the local lymph nodes because a large proportion of particles of this size will impact on the mucous membrane during passage of inhaled air through the nose.

Small quantities of inhalant allergen cause immediate hypersensitivity

Estimates of the quantity of mite or pollen-derived proteins inhaled vary from 5–50 ng/day. Thus exposure to some allergens may be as little as 1 μg/year. This is important because it probably explains:

• why the immune response is consistently of this one kind (i.e. immediate hypersensitivity); and

• why no respiratory diseases, other than asthma, have been associated with these allergens.

The quantities inhaled also seriously restrict the models about how allergens contribute to asthma. Inhaling a small number (i.e. 10–100) of ‘large’ particles (10–30 μm in diameter) per day could produce local areas of inflammation but would not be expected give rise to alveolitis, acute bronchospasm, or progressive lung fibrosis.

Only a small number of food proteins are common causes of allergic responses

Although many food proteins can occasionally give rise to IgE responses, only a small number are common causes of food allergy. These include egg, milk, wheat, soy, tree nuts, peanut, fish, and shellfish. In contrast to inhaled allergens, these proteins are often eaten in very large quantities (i.e. ≈10–100 g/day). In general only a small fraction of these food proteins is absorbed. However, small peptides can be freely absorbed and may be recognized by T cells and even by IgE antibodies in a minority of individuals. Nevertheless, the bulk of the allergic and anaphylactic responses to foods are thought to be related to food proteins that have not been digested, either triggering mast cells in the intestine or entering the circulation.

Q. Recent evidence from Dr Sampson and his colleagues has shown that some children produce IgE antibodies against linear epitopes on food allergens. What implication does this have for induction of allergy in cooked food?

A. Denaturation of a protein by heat will not destroy linear epitopes, consequently cooking will not alter the allergenicity.

IgE binding sites can be identified on the tertiary structure of allergens

Many different allergens have been cloned, and for a few the tertiary structure is now known either from X-ray crystallography (e.g. Bet v1), by nuclear magnetic resonance (e.g. Der p2), or by modeling relationships to known homologs (Fig. 23.w1).

Fig. 23.w1 Tertiary structure of two important allergens – Der p2 and Bla g2

The tertiary structure of the dust mite allergen, Der p2 (1), was derived by nuclear magnetic resonance. This protein has no known function, and the structure shows no enzymic sites. On this structure the amino acids that are part of the binding sites for two monoclonal antibodies are shown (DPX green and 7A1 red). Knowledge of the structure makes it possible to alter the antibody binding sites using site-directed mutagenesis. (2) The tertiary structure of a cockroach allergen, Bla g2, shows the enzymic cleft and three residues that are considered to form the active site of an aspartic protease. However, the molecule has no enzymic activity due to other changes in the primary amino acid sequence. Some allergens (e.g. Der p1 and Blag 5) are active enzymes, but it remains an open question whether this activity contributes to their activity as allergens. On each structure the β sheets are shown as green bands, the α-helical structures in Bla g2 are shown in red. In addition, cysteines are shown as yellow spheres.

((1) Courtesy of Drs Alisa Smith and Geoffrey Mueller. (2) Courtesy of Drs Anna Pomes and Martin Chapman.)

Knowledge of the tertiary structure makes it possible to predict surface residues and to define IgE binding sites using site-directed mutagenesis. This approach provides the potential to design molecules that have decreased IgE binding properties, but with preserved T cell epitopes.

Desensitization can be used to control type I hypersensitivity

Given the importance of T cells to the control of IgE antibody production and their potential role in the recruitment of inflammatory cells, it is logical to try treatments which directly ‘desensitize’ T cells. The approaches used include treatments with modified allergens, including:

• allergen molecules modified in vitro by formaldehyde or glutaraldehyde (allergoids);

• site-directed mutagenesis;

• allergens combined with two to four molecules of CpG;

• peptides of 12–35 amino acids.

Therapeutic trials have been carried out with peptides from ragweed pollen antigens and the cat allergen Fel d1. The results show that peptide recognition is restricted by the HLA-DR type of the patient, which means that a wide range of peptides are necessary for treatment. In addition, there is clear evidence that peptides can produce a significant response in the lungs (Fig. 23.9) indicating that T cells in the lung can contribute to an asthmatic response.

Fig. 23.9 Late asthmatic response to peptides from cat allergen

Late asthmatic reactions induced in cat allergic patients by the intradermal injection of peptides derived from the cat allergen, Fel d1. The nine responders show a mean fall in forced expiratory volume in 1 second (FEV₁) of approximately 30%. The response to the peptides is MHC-restricted and correlated with the ability of the patients’ T cells to respond to these peptides in vitro. On challenge days (red filled circle) injection of peptides was associated with a fall in FEV₁ which did not occur on the control days (black filled circle). Data are shown for nine responders (upper graph) and 31 non-responders (lower graph).

(Courtesy of Dr Mark Larché from J Exp Med 1999;189:1885.)

Mediators released by mast cells and basophils

The only human cell types that contain histamine are mast cells and basophils. In addition, these are the only cells that express the high-affinity receptor for IgE (FcεRI) under resting conditions.

The primary and most rapid consequence of allergen exposure in an allergic individual is cross-linking of IgE receptors on mast cells and basophils:

• basophils are circulating polymorphonuclear leucocytes that are not present in normal tissue, but can be recruited to a local site by cytokines released from either T cells or mast cells;

• mast cells cannot be identified in the circulation, but are present in connective tissue and at mucosal surfaces throughout the body.

Mast cells in different tissues are morphologically and cytogenetically distinct.

Both the cells that contain histamine and the biology of these cells may be very different in other species. For example:

• in the rabbit the histamine content of the peripheral blood is almost all in platelets;

• in the mouse there are few if any circulating basophils; and

• in rats the degranulation of mast cells appears to be one granule at a time.

By contrast, in human mast cells and basophils the granules fuse with the exterior membrane and release their contents as a solution. The membrane of the granule then becomes part of the plasma membrane (Fig. 23.10).

Fig. 23.10 Human basophils

Basophils are circulating mononuclear cells that have multilobed nuclei and distinctive granules that stain with metachromatic stains (1). Basophils can be recruited into local tissues such as the skin, nose, lungs, or gut by allergic and other immune responses. (2) A basophil degranulating 4 minutes after adding allergen. The degranulation that releases histamine occurs by fusion of the granule membrane with the external membrane of the cell. (C, centriole; cp, coated pit; er, endoplasmic reticulum; np, nuclear pore; G, Golgi apparatus; lv, lucent vesicle; m, mitochondria; ng, nuclear granule; rg, residual material from granules; sg, small granules.)

(Courtesy of Robin Hastie.)

Mast cells in different tissues have distinct granule proteases

Mast cells were originally identified by Ehrlich who named them based on the distinctive, tightly packed granules. (Mast means well fed, or fattening, in German.) Mast cells in different tissues can be distinguished by staining for proteases, and the content of these enzymes may be relevant to their role in allergic diseases. The granule proteases of mast cells have been cloned and sequenced and are distinct for two types of mast cell (Fig. 23.11):

• mucosal mast cells are characterized by the presence of tryptase without chymase (MCT);

• by contrast, connective tissue mast cells contain both chymase and tryptase (MCTC).

Fig. 23.11 Differences between mast cell populations

(1) There are at least two subpopulations of mast cell, the mucosal mast cells (MMCs) and the connective tissue mast cells (CTMCs). The differences in their morphology and pharmacology suggest different functional roles in vivo. MMCs are associated with parasitic worm infections and, possibly, allergic reactions. In contrast to the CTMC, the MMC is smaller, shorter lived, T cell dependent, has more Fcε receptors, and contains intracytoplasmic IgE. Both cells contain histamine and serotonin in their granules; the higher histamine content of the CTMC may be accounted for by the greater number of granules. Major arachidonic acid (AA) metabolites (prostaglandins [PGs] and leukotrienes [LTs]) are produced by both mast cell types, but in different amounts. For example, the ratios of production of the leukotriene LTC₄ to the prostaglandin PGD₂ are 25:1 in the MMC and 1:40 in the CTMC. The effect of drugs on degranulation is different between the two cell types. Sodium cromoglycate (DSCG) and theophylline both inhibit histamine release from the CTMC, but not from the MMC. (This may have important implications in the treatment of asthma.) Note that some of these data come from rodent studies and may not apply to humans. (2) Tryptase is a tetramer of 134 kDa that may comprise as much as 25% of the mast cell protein. Chymase is a monomer of 30 kDa. The relative proportions of these proteases in mast cells define MC_T and MC_TC populations, which have different distributions in human tissues. Basophils have very low amounts of both proteases. (The suffixes T and TC represent tryptase and chymase present in the respective cells.)

These enzymes may play a direct role in the lung inflammation of asthma, either by breaking down mediators or, in the case of tryptase, by acting as a fibroblast growth factor. Basophils contain very little of either of these proteases.

Staining of basophils in tissue sections requires special fixation and staining. Without this staining the granules in basophils cannot be identified and the cells appear as neutrophils (i.e. polymorphonuclear cells without eosinophilic or basophilic granules).

Cross-linking of FcεRI receptors results in degranulation

The process of degranulation in human mast cells and basophils involves fusing of the membrane of the granules containing histamine with the plasma membrane (see Fig. 23.10). The granule contents rapidly dissolve and are secreted, leaving behind a viable degranulated or partially degranulated cell. This process is initiated in most cases by cross-linking of two specific IgE molecules by their relevant allergen.

When two IgE receptors (FcεRI) are cross-linked, signal transduction through the γ chains of the receptor (see Fig. 23.12) leads to influx of calcium, which initiates both degranulation and the synthesis of newly formed mediators (Fig. 23.12).

Fig. 23.12 Mast cell mediator release

Mast cells release mediators after cross-linking of the IgE receptors on their surface. Preformed mediators are released rapidly while arachidonic acid metabolites such as leukotriene D₄ and prostaglandin D₂ are released more slowly. Mast cells can also be triggered by opiates, contrast media, vancomycin, and the complement components C3a and C5a. The mediators, which are also released by basophils, include histamine, TNFα, and IL-4. Histamine released by mast cells can be measured in serum following anaphylaxis or extensive urticaria, but it has a half-life in minutes. By contrast, tryptase can be measured in serum for many hours after an anaphylactic reaction.

Other mechanisms can be involved. Experimentally, degranulation can be triggered through FcεRI by using:

• anti-IgE;

• lectins such as phytohemagglutinin (PHA) or concanavalin A (Con A); or

• formyl-met-leu-phe (FMLP).

Q. Give an example of a mediator that can cause mast cell degranulation without cross-linking FcεRI

A. Typical examples include the complement components C5a and C3a (see Fig. 6.18).

Drugs such as codeine or morphine, the antibiotic vancomycin, and contrast media used for imaging the kidneys also degranulate mast cells. Acute reactions to these agents, which are not thought to involve IgE antibodies, are referred to as anaphylactoid.

In allergic individuals mast cells can be recruited to the skin and to the nose

Although mast cells are present in normal non-inflamed tissue, their numbers are increased in response to inflammation. It is assumed that this accumulation is T cell dependent because in rats infected with Nippostrongylus brasiliensis accumulation of mast cells in the gut is dependent on T cells and can be suppressed by corticosteroids.

In guinea pigs the immune response to tick bites includes a large local accumulation of basophils. Indeed, the tick is thought to be killed by basophils that it ingests.

In allergic individuals mast cell recruitment has been demonstrated both:

• in the skin in response to repeated allergen exposure; and

• in the nose during the pollen season. In both situations basophils are also recruited.

In the nose the recruitment of cells represents a shift so that mast cells move from the subepithelium into the epithelium while basophils appear in the nasal mucus. This process, which brings histamine-containing cells closer to the site of entry of allergen, is one of the ways in which allergic individuals become more sensitive. It is likely but less well established that equivalent processes occur in the human lung and gut.

Genetic associations with asthma

Hayfever, asthma, and atopic dermatitis are common in allergic families. So that children with one allergic parent have a 30% chance of developing allergic disease; those who have two allergic parents have as high as a 50% chance.

Systematic studies of allergic diseases are complicated because the phenotypes for diseases, such as hayfever and asthma, are not well defined and depend on the approach used to make the diagnosis. Although on average, total IgE values increase progressively from normal, in hay fever, asthma, and atopic dermatitis, the individual values vary widely (Fig. 23.13).

Fig. 23.13 IgE levels and atopic disease

(1) The serum concentration of IgE (which is around 100 IU/mL) is only approximately 0.001% that of IgG (around 10 mg/mL) and comprises less than 0.001% of the total immunoglobulin. Levels in atopic patients tend to be raised, and this is especially so in atopic eczema (the International Unit [IU] for IgE equals 2.4 ng). (2) The higher the level of IgE, the smaller the percentage of the population is affected, but the greater the likelihood of atopy. Where the level is greater than 450 IU/mL the majority of subjects are atopic.

Asthma defined by a patient questionnaire is therefore less specific than asthma defined by testing of specific or non-specific bronchial hyperreactivity. Furthermore, studies on asthma are complicated because several aspects are under genetic control, including

• IgE antibody responses;

• the inflammatory response to allergens;

• repair mechanisms; and

• bronchial reactivity.

Indeed, it is important not to confuse simple genetic diseases like cystic fibrosis or hemophilia with complex traits such as asthma or type II diabetes mellitus.

It is therefore not surprising that multiple genes (currently at least 50), have been associated with asthma in different populations.

A further major problem in interpreting genetic analyses of allergic disease comes from the progressive increase in the incidence of asthma between 1960 and 2000. Clearly this increase cannot be attributed to genetic change and implies that some of the genes identified would influence asthma only in the presence of other changes either in the environment or in lifestyle. This is referred to as a gene–environment interaction.

The genetics of asthma has been studied both by genomic screening and by using candidate genes. Genomic screening identifies regions of the genome that link to asthma so that this region can be examined to identify specific genes.

If a candidate gene is identified, it is possible to examine the gene for polymorphisms that link to asthma. However, a brief consideration of the possible targets (Fig. 23.14) makes it clear how complex the analysis of asthma is likely to be, and indeed is proving to be. Typical examples include polymorphisms of the promoter region for IL-4 and polymorphisms of the gene for IL-5.

Fig. 23.14 Genetic influences over asthma and allergic diseases

Allergic diseases run in families, but the inheritance is not simple. Population-based studies have established that the inheritance of allergic diseases is influenced by multiple genes. Some of these, such as HLA-linked control of the response to pollen antigens or genes controlling total IgE, are related to the immune response. However, many others are related to the mechanisms of inflammation (e.g. IL-4 and IL-5 gene polymorphisms) or to the response to treatment (e.g. leukotriene receptor genes or polymorphisms of the β₂-adrenergic receptor).

Q. What effect might polymorphism in these genes have?

A. The level of production of IL-4, and the activity of IL-5, will vary between individuals, each of which will affect the type of immune response that develops – TH1 versus TH2 – either of which could directly influence the inflammatory response that occurs as a result of exposure to allergens.

A further series of polymorphisms have been identified that influence the response of asthma to treatment. These include:

• variants of the β₂-adrenergic receptor α chain; and

• genetic differences that influence the therapeutic response to leukotriene antagonists.

At present, it appears that the overall effects are too complex to be of any practical significance. Certainly it is most unlikely that gene transfer will ever be of significance therapeutically. However, as genetic screening becomes easier, pharmacogenetics may become an important method for identifying the best drugs for individual patients.

Skin tests for diagnosis and to guide treatment

The primary method for diagnosing immediate hypersensitivity is skin testing. The characteristic response is a wheal and flare (Figs 23.15 and 23.16):

• the wheal is caused by extravasation of serum from capillaries in the skin, which occurs as a direct effect of histamine and is accompanied by pruritus (also a direct effect of histamine);

• the larger erythematous flare is mediated by an axon reflex.

Fig. 23.15 Skin tests

Skin tests are carried out by introducing 0.02 mL of extract intradermally. With allergens such as pollen, cat, or dust mite, the positive reaction is an immediate (i.e. within 20 min) wheal, which in some cases is followed by an indurated response occurring late (i.e. at 4–12 hours). Non-allergic individuals make no discernible reaction to testing with these allergens. A delayed skin response is the commonest form of positive response to tuberculin, tetanus, and mumps, or to fungi such as Trichophyton and Candida spp. The skin typically shows no reaction up to 12 hours and then gradually develops an erythematous, indurated, delayed hypersensitivity response, which is maximal at 24–48 hours. Patch tests are performed by applying a gauze pad with allergen to a patch of skin that has been mildly abraded. This procedure may give an immediate wheal response, but this is followed at 24–48 hours by an indurated, erythematous response, which has many of the features of eczema. The patch test is not a diagnostic test, but has provided extensive information about the role of allergens in atopic dermatitis.

Fig. 23.16 Skin test reactions

(1) A type-I hypersensitivity reaction produces a raised wheal 5–7 mm in diameter and with a well-defined edge after about 15 minutes. (2) An erythematous and eczematous skin response 48 hours after the application of 5 μg of the mite allergen Der p1 to the skin of a patient with atopic dermatitis who had 56 IU/mL of IgE antibody to the dust mite Dermatophagoides pteronyssinus. Biopsy of the patch site revealed an infiltrate of eosinophils, basophils, and lymphocytes.

This skin response takes 5–15 minutes to develop and may persist for 30 minutes or more. Techniques for skin testing include:

• a prick test, in which a 25-gauge needle or a lancet is used to introduce ~0.2 μL of extract into the dermis;

• an intradermal injection of 0.02–0.03 mL.

All allergen injections have the potential to cause anaphylaxis and for safety reasons the intradermal test, which introduces approximately 100 times more extract, should always be preceded by a prick test.

Skin tests are evaluated by the size of the wheal compared to a positive (histamine) and negative (saline) control. In general, a 3 × 3 mm wheal in children and a 4 × 4 mm wheal in adults can be considered a positive response to a prick test.

A positive skin test indicates that the patient has specific IgE antibodies on the mast cells in their skin. In turn this implies that bronchial or nasal challenge would also be positive if sufficient antigen were administered.

In most cases (i.e. ≈80%) where the skin prick test is positive, IgE antibody will be detectable in the serum. However, blood tests for IgE antibody are generally less sensitive than intradermal skin tests.

Q. Suggest explanations for the higher sensitivity of the skin test

A. Most IgE is located on the high-affinity receptor on mast cells. Its half-life is 10 days on the mast cell as opposed to 2 days in blood. Sensitized mast cells can react to concentrations of allergen as low as 10⁻⁵ micrograms.

Positive skin tests are common

Epidemiologically, sensitization to a relevant inhalant allergen is a ‘risk factor’ for allergic disease. An individual with a positive skin test to grass pollen is therefore up to ten times more likely to have hayfever during the grass pollen season (odds ratio ≥10) than a skin test-negative individual. Equally an individual with a positive skin test to dust mite or cat allergen is more likely to have asthma (odds ratios 2.0 to >6.0). It is assumed that allergen exposure contributes to the risk, but that the relationship is not simple.

Positive skin tests are common, and in individual cases may not be relevant, because the patient is not exposed to the allergen, for example:

• grass pollen is not relevant to understanding symptoms occurring during autumn;

• equally skin tests to cat dander or cockroach allergens may not be relevant if the patient has moved to an area or house where those allergens are not present.

In addition, up to one-third of skin test-positive individuals do not experience symptoms when they are exposed to the relevant allergen.

Following a strongly positive skin test, the skin response may return at 6–12 hours as an indurated late response which involves both the prolonged effects of mediators and a cellular influx.

Late skin reactions probably include several different events

Late reactions can occur following an immediate response to allergen, in either the skin or the lungs.

A late skin response is only common following a large immediate response (i.e. wheal size 10 × 10 mm). The late response, which is diffuse, erythematous, and indurated, generally starts 2–3 hours after the wheal and may last for up to 24 hours. The late reaction is considered to be a model of the events that lead to persistent inflammation in the nose, lungs, or skin.

Late reactions probably include several different events:

• the direct effects of prostaglandins, leukotrienes, and cytokines released by mast cells following the initial release of histamine;

• infiltration of lymphocytes, eosinophils, basophils, and neutrophils into the local site mediated by chemokines and other cytokines released from mast cells;

• release of products from the infiltrating cells. In general, these events are occurring in parallel over a period of hours.

True delayed skin test responses (i.e. without an immediate response) are:

• characteristic of the response to tuberculin; and

• common with fungal antigens, particularly to the yeast Candida albicans or the dermatophyte fungus Trichophyton spp.

By contrast true delayed responses are very rare following skin testing with pollen, animal dander, or dust mites.

Pathways that contribute to the chronicity of allergic diseases

The release of histamine that occurs within 15 minutes after allergen exposure can only explain a proportion of allergic disease. The chronic inflammation in the lungs of patients with asthma and in the skin of patients with atopic dermatitis cannot be explained by histamine because:

• the time course is too long;

• there is a cellular infiltrate in these tissues; and

• there are major differences in disease between patients who have apparently similar titer and specificity of IgE in their serum.

Several different pathways contribute to chronic symptoms and can alter the severity or chronicity of allergic disease.

• Local recruitment of mast cells and basophils, combined with increased ‘releasability’ of these cells, allows an increased response to the same allergen challenge – this mechanism plays a major role in the increased symptoms in the nose during the pollen seaseon.

• Release of leukotrienes, chemokines and cytokines from mast cells, or basophils; these mediators can have direct effects on blood vessels and smooth muscles. In addition IL-5, tumor necrosis factor (TNFα), and chemokines are each thought to contribute to the recruitment of inflammatory cells.

• T cells can be recruited to local tissues and can release a wide range of cytokines which have direct inflammatory effects.

Atopic dermatitis and the atopy patch test

Patients with atopic dermatitis (AD) have the highest levels of both specific IgE and total IgE. Thus IgE antibodies of ≥100 IU/mL (class 6), specific for dust mite, cockroach, pollens, or fungi, are common. Equally total IgE levels in patients with severe AD are usually ≥2000 IU/mL. However, there are still major disagreements about the importance of allergen exposure to the symptoms of this disease. This is because:

• the time course of the disease is chronic;

• injection of allergen into the skin causes a wheal and flare response, and doesn’t consistently cause eczema;

• the disease is multi-factorial, including a role for food allergy, skin infection, genetic variations in skin barrier function (based on filaggrin), and also inhalant allergens.

The atopy patch test provides an important model of the ways in which allergen applied to the skin can induce eczema.

Epidermal spongiosis and a dermal infiltrate are features of a positive patch test

The infiltration of cells into the skin that occurs in the 24 hours after an allergen is applied can be studied in several ways:

• by local intradermal injections;

• by applying a patch of allergen on gauze that stays on the skin for 2 days; or

• by fixing a chamber containing allergen over a denuded area of skin.

The skin chamber allows repeated sampling whereas the other two techniques require biopsy of the skin.

In the patch test 10 μg allergen is applied on a gauze pad 2.5 cm², and the biopsy is carried out at 24 or 48 hours. A positive patch response induces:

• macroscopic eczema;

• spongiosis of the epidermis (a hallmark of eczema); and

• an infiltrate of cells into the dermis (see Fig. 23.16).

The cellular infiltrate includes eosinophils, basophils, and lymphocytes.

With persistent allergen at a site (i.e. 6 days), the eosinophils degranulate locally. This is in keeping with the evidence that the skin of patients with eczema contains large quantities of the eosinophil granule major basic protein (MBP), even though very few whole eosinophilic cells are visible (Fig. 23.17).

Fig. 23.17 Eosinophil major basic protein in the skin of atopic dermatitis

Skin biopsy from a patient with severe atopic dermatitis. The hematoxylin and eosin (H&E) stain (1) shows an inflammatory infiltrate, but very few intact eosinophils are present. The same section stained with antibodies to eosinophil major basic protein (MBP) (2) shows extensive deposition of MBP in the dermis, demonstrating that eosinophils had degranulated in the skin.

(Courtesy of Dr K Lieferman.)

Biopsy of patch tests also yields T cells that are specific for the allergen used, which in most cases has been dust mite, thus establishing that antigen-specific T cells are present in the skin after antigen challenge.

Answering whether allergen-specific T cells are present at local sites is important because T cells could play a role both as effector cells and in the recruitment of other cells.

Establishing whether T cells play an effector role is also relevant to the nose in rhinitis, the lungs in asthma, the conjunctiva in hayfever, as well as the skin in atopic dermatitis.

Biopsy of patch test sites has also established that the Langerhans’ cells in the skin of patients with eczema express FcεRI. It is assumed that these cells use IgE antibodies to help capture allergens and to increase the efficiency of antigen presentation.

Therefore, in any analysis of the factors influencing the severity of allergic disease (e.g. response to pharmacological treatment or response to immunotherapy), it is necessary to consider the relevance of both mast cells and effector T cells.

Allergens contribute to asthma

The causal role of bee venom in anaphylaxis or grass pollen in seasonal hayfever is obvious because:

• these diseases occur in individuals who have positive skin tests; and

• the symptoms are directly related to increased exposure.

By contrast, the role of inhaled allergens in chronic asthma is less obvious because exposure is perennial, the patients are often not aware of the relationship, and only a proportion of skin test-positive individuals develop asthma.

The evidence that allergens derived from dust mites, cats, dogs, the German cockroach, or the fungus Alternaria spp. contribute to asthma comes from several different lines of evidence:

• the epidemiological evidence that positive skin tests or serum IgE antibodies are a major risk factor for asthma;

• bronchial challenge with nebulized extracts can produce both rapid bronchospasm, within 20 minutes, and a late reaction in 4–8 hours, which is characterized by renewed mediator production and a cellular infiltrate;

• reduced exposure to allergens can lead to decreased symptoms and decreased non-specific bronchial reactivity – this avoidance can be achieved either by moving patients to an allergen-free unit or by controlling exposure in the home.

Bronchi in the lungs of patients with asthma are characterized by increased mast cells, lymphocytes of the TH2 type, eosinophils, and products of eosinophils. In addition, there is increased mucus production secondary to goblet cell hyperplasia, epithelial desquamation, and collagen deposition below the basement membrane. These changes are a reflection of chronic inflammation, and it is generally considered that eosinophils play a major role in these events (Fig. 23.18).

Fig. 23.18 Inflammatory response in asthmatic bronchi

Mast cells release factors that can induce immediate bronchospasm (e.g. histamine and LTD₄), but also release chemotactic factors such as LTB₄, IL-5, and TNFα. The spasmogens can induce edema, increased mucus, and smooth muscle constriction, resulting in an immediate decrease in airway conductance and a fall in FEV₁. By contrast, chemotactic factors recruit cells out of the circulation including eosinophils, neutrophils, lymphocytes, and macrophages. These cells can chronically modify the lung with goblet cell hyperplasia, collagen deposition below the basement membrane, and possibly smooth muscle hyperplasia. In addition, these cells and their products produce non-specific bronchial hyperreactivity. Thus, chronic bronchospasm includes elements of hypersecretion, inflammatory infiltrate, thickening of the walls of the small bronchi, and bronchial smooth muscle spasm. Evidence for this inflammatory response can be obtained from increased exhaled nitric oxide; increased eosinophils or eosinophil cationic protein (ECP) in induced sputum; and experimentally from biopsies of the lung. (1, neutrophils; 2, basophils; 3, eosinophils; 4, monocytes; C, cells; M, mucus; SM, smooth muscle.)

Q. Some recent evidence has shown that anti-IL-5 treatment has limited effects on asthma, though it decreases circulating eosinophils. How do you interpret this result?

A. In part this can be explained because other cells contribute in the inflammation in asthma, however, in addition eosinophils in the nases can be recruited and maintained by cytokines other than IL-5.

BAL analysis after allergen challenge demonstrates mast cell and eosinophil products

Analysis of bronchoalveolar lavage (BAL) after an allergen challenge demonstrates the presence of products derived from mast cells and eosinophils.

Q. What products would you expect to detect in BAL following challenge?

A. Histamine, prostaglandins, and leukotrienes from mast cells; major basic protein (MBP) and eosinophil cationic protein (ECP) from eosinophils (see Chapter 2).

Furthermore, MBP is present in biopsies of the lungs and can produce epithelial change typical of asthma in vitro (Fig. 23.19).

Fig. 23.19 Localization of MBP in the lung of a severe asthmatic

(1) Respiratory epithelium showing striking submucosal eosinophil infiltration and a cluster of desquamated epithelial cells in the bronchial lumen (arrowed) next to a ‘stringy’ deposit of soot. H&E stain. (2) The same section stained for major basic protein (MBP) showing immunofluorescent localization in infiltrating eosinophils. MBP deposits are also seen on desquamated epithelial cells on the luminal surface. (3) A control section stained with normal rabbit serum does not stain eosinophils or bronchial tissue, but does show some non-specific staining of the sooty deposit.

(Courtesy of Dr G Gleich, reprinted from J. Allergy Clin. Immunol. 1982;70:160–169, with permission from American Academy of Allergy Asthma and Immunology.)

The subepithelial collagen deposition present in many patients with asthma is probably a reflection of fibroblast responses to local inflammation.

Although it has been suggested that these changes, which are referred to as ‘remodeling’, can lead to progressive decreases in lung function, the evidence for this view is not clear. In particular, progressive loss of lung function is unusual in asthma and there are no studies showing a correlation between the extent of collagen deposition and changes in lung function. Nonetheless, inhaled corticosteroids, which can block many different aspects of inflammation, are an effective long-term treatment that can control asthma. The effects of corticosteroids include:

• blocking the delayed response in the lungs;

• inhibiting influx of eosinophils, basophils, and lymphocytes;

• reducing eosinphil production in the bone marrow;

• inhibiting transcription of genes for IL-5, TNFα, and some chemokines;

• reducing T cell activity.

Locally active corticosteroids are widely used in seasonal rhinitis, perennial rhinitis, asthma, and atopic dermatitis. In addition, courses of systemic corticosteroids are used for the treatment of exacerbations of asthma.

Bronchial hyperreactivity is a major feature of asthma

Non-specific bronchial hyperreactivity (BHR) is present in patients with asthma and is a major feature of the disease. Thus, airway obstruction, induced by cold air or exercise, and nocturnal asthma all correlate with non-specific bronchial reactivity. BHR can be demonstrated by challenging the lungs with histamine, methacholine, or cold air.

The mechanism by which exercise or cold air induces a bronchial response is thought to be evaporation of water with associated cooling of the epithelium. However, it is unclear whether this process triggers nerve endings directly or by causing local mediator release.

Evidence for inflammation of the lungs of patients with asthma is indirect

Bronchoscopy is not possible in patients with asthma except as a research procedure. Therefore the only evidence for inflammation of the lungs that can be obtained routinely is indirect:

• peripheral blood or nasal smear eosinophils are increased in most patients presenting with an acute episode of asthma (Fig. 23.20);

• nasal secretions may contain increased ECP and IL-8 (CXCL8).

Fig. 23.20 Nasal eosinophils

Nasal smear from an 8-year-old boy presenting with acute asthma. Most of the cells are eosinophils – polymorphonuclear cells with a cytoplasm that stains red using H&E stain. He was known to be allergic to dust mites and had recently had a rhinovirus infection as judged by polymerase chain reaction on nasal secretions.

Additional evidence about inflammation in the lungs can be obtained either from exhaled air or from condensates of exhaled air. Nitric oxide (NO^•) gas is increased in patients with asthma, and this decreases following systemic or local corticosteroid treatment.

Q. Why should NO^• be increased in the lungs of asthmatic patients?

A. Macrophage activation causes synthesis of inducible nitric oxide synthase (iNOS) and consequent NO^• production in the lungs (see Fig. 7.19).

In addition, the pH of the condensate decreases during acute episodes. The increased exhaled NO^• may reflect upregulation of the enzyme iNOS. In many studies exhaled NO appears to be closely related to allergic inflamation. In adults further information about the inflammation in the ‘respiratory tract’ can be obtained from computed tomography (CT) of the nasal sinuses. Extensive opacification of the sinuses is present in approximately one-third of patients presenting with acute asthma. This reflects both:

• chronic sinusitis, which is a major feature of late-onset asthma; and

• sinus inflammation secondary to acute rhinovirus infection.

Whether the changes in the sinuses are a reflection of similar effects occurring in the lungs, or a source of mediators, or T cells that contribute to lung inflammation, is not clear.

Treatments for type I hypersensitivity

Immunotherapy is an effective treatment for hayfever and anaphylactic sensitivity to venom

Immunotherapy (or hyposensitization) with allergen extracts was introduced in 1911 by Noon and Freeman. At that time they were trying to establish immunity against pollen toxin.

Immunotherapy requires regular injections of allergen over a period of months. It is an established treatment for:

• seasonal hayfever; and

• anaphylactic sensitivity to bees, wasps, and hornets.

In addition, immunotherapy is an effective treatment for selected cases of other allergic diseases including asthma.

The dose is increased progressively, starting with between 1–10 ng and increasing up to approximately 10 μg allergen per dose.

The response to treatment includes:

• an increase in serum IgG antibodies;

• a striking decrease in the response of peripheral blood T cells to antigen in vitro; and

• a marked decrease in late reactions in the skin.

Over a longer period of time there is a progressive decrease in IgE antibodies in the serum (Fig. 23.21).

Fig. 23.21 Effects of immunotherapy on allergic rhinitis

During desensitization or immunotherapy the allergic patient receives regular subcutaneous injections of the relevant allergen. The immunological changes that occur include an initial increase in IgE antibodies followed by a gradual decline, which in pollen-allergic patients is largely due to a blunting of the seasonal increase. Antibodies of the IgG and specifically IgG4 isotype increase progressively and may reach concentrations of ten times those present before treatment. Symptoms decline, starting as early as 3 months, but generally not maximally until 2 years. Changes in T cells are less well defined, but include decreased in-vitro response to allergens and increased production of IL-10.

The change in antibodies, lymphocyte responses, and symptoms could all be secondary to changes in T cells. Given the known mechanisms of allergic inflammation, a response of T cells to allergen injections could influence symptoms in several ways:

• decreased local recruitment of mast cells and basophils;

• decreased recruitment of eosinophils to the nose or lungs;

• increased IgG including IgG4 antibodies with progressive decreases in IgE – the IgG antibodies may act as blocking antibodies by binding allergen before it cross-links IgE on mast cells.

Some studies of cytokine RNA have suggested that immunotherapy produces a shift in T cells from a TH2 profile (i.e. IL-4 and IL-5) towards a profile that is more typical of TH1 (i.e. IFNγ). Although this could explain decreased help for IgE, and decreased eosinophil recruitment, this would not explain the production of IgG4. The expression of the gene for IgG4 is dependent on IL-4, and may also require the cytokine IL-10. The response to immunotherapy is therefore better seen as a modification of the TH2 response.

Modified forms of allergen-specific immunotherapy

Peptides from the primary sequence of an allergen that can stimulate T cells in vitro

Peptides from the primary sequence of an allergen, usually approximately 20 amino acids in length, stimulate T cells in vitro and in vivo.

In theory, peptides provide a mechanism for stimulating or desensitizing T cells without the risk of anaphylaxis, which is always present with traditional allergens.

Whether incomplete stimulation of T cells by peptides can lead to ‘tolerance’ or a change in the cytokine profile is not clear. Problems with peptide immunotherapy include:

• significant reactions in the lung with a fall in FEV₁; and

• the fact that multiple peptides are necessary to allow presentation of antigen in patients with different HLA types.

Modified recombinant allergens have decreased binding to IgE

Genetically modified recombinant allergens that have decreased binding to IgE can be produced. Their advantage is that the primary sequence with the T cell epitopes is preserved. Even if the molecule is extensively modified, any full-length protein has the potential to induce anaphylaxis in allergic individuals. Thus, the use of genetically modified molecules would always require precautions similar to those for traditional immunotherapy. A potential but unlikely problem is that patients would develop IgE antibodies against new epitopes.

Adjuvants can shift the immune response away from a simple TH2 response

Adjuvants ‘attached’ to allergen molecules have been designed to shift the immune response from TH2 towards TH1. Possible co-molecules that act like an adjuvant include:

• the cytokine IL-12; or

• immunostimulatory sequences (ISSs). ISSs are DNA sequences such as cytosine phosphoguanidine (CpG) that are common in bacterial DNA and have a profound effect on the mammalian immune system.

In mice combining an antigen with two or three molecules of CpG can induce a TH1 response or downregulate IgE responses.

Combining CpG with allergen not only influences the response, but also reduces the reactivity of the allergen with IgE.

Thus immunization with allergen and CpG may produce a greater immune response with less potential for an acute allergic reaction.

Although preliminary trials with CpG were encouraging large trials were not. It is important to remember that CpG acts through TLR9 which is on the nuclear membrane. An alternative approach is to use flagellin attached to allergen because it binds to TLR-5 which is on the cell surface.

DNA vaccines are being designed to change the immune response

The concept of immunizing with the gene for an antigen is well established (i.e. DNA vaccines). Experiments with DNA vaccines have been very successful in mice, both in inducing a TH1 response initially and in controlling an existing IgE antibody response. However, the consequences of expressing an allergen within the tissue of an allergic individual are not known. Equally, it is not clear whether inducing a TH1 response to a high dose allergen such as cat dander which is present in almost all houses would give rise to other forms of inflammatory disease.

Other forms of immune based non-specific therapy

Humanized monoclonal anti-IgE

Humanized monoclonal anti-IgE treatment can reduce sensitivity and significantly decrease the number of acute episodes of asthma per year.

Antibodies directed against the binding site for FcεRI on IgE bind to IgE in the circulation, but not when it is attached to mast cells or basophils. An antibody of this kind can therefore remove IgE from the circulation but will not induce anaphylaxis. A mouse monoclonal antibody to IgE has been progressively humanized so that the molecule can be safely injected into patients and will bind IgE with high affinity.

Treatment with anti-IgE antibodies has reduced exacerbations of asthma and the symptoms of hayfever. In addition, continued treatment that controls free IgE below 10 ng/mL leads to a progressive decrease in the number of IgE receptors on mast cells. Thus, the treatment may achieve a secondary effect, further decreasing the sensitivity of histamine-containing cells to allergen. Although there have been small studies on the role of anti-IgE in treating food allergy, atopic dermatitis, urticaria and drug allergy, this treatment remains to be established.

Recombinant soluble IL-4R can block the biological activity of IL-4

Given the central role of IL-4 in the TH2 response, it is not surprising that several efforts have been made to block its action. These include:

• a mutated IL-4 (Y124D);

• antibodies to IL-4; and

• recombinant soluble IL-4 receptor (sIL-4R). Treatment with sIL-4R has proved moderately effective in clinical trials of allergic asthma. The mechanism is that sIL-4R binds to IL-4 before it can react with the receptor on T cells or B cells, and thus blocks its biological activity. However, it is less clear which of the many actions of IL-4 is relevant to the clinical effects:

• blocking the action of IL-4 on B cells may reduce IgE production, but this would probably require many weeks to produce a clinical effect; because IgE plasma cells are long lived;

• the autocrine effect of IL-4 on TH2 cells may be an essential growth factor.

The efficacy of sIL-4R provides indirect evidence for the role of T cells in allergic disease.

Humanized monoclonal anti-IL-5 decreases circulating eosinophils

Anti-IL-5 (like anti-IgE) is a humanized mouse monoclonal antibody.

Following successful studies in baboons, anti-IL-5 has been shown to decrease circulating eosinophils in patients. It is therefore assumed that binding IL-5 produced by T cells (or mast cells) can decrease the production of eosinophils in the bone marrow. However, the results do not answer whether the treatment acts on IL-5 in the circulation or on IL-5 produced by T cells (or mast cells) locally in the bone marrow and/or the respiratory tract.

In initial studies, anti-IL-5 was not effective as a treatment for asthma despite an approximately 90% decrease in circulating eosinophils.

Some new treatment approaches may not be practical

The primary treatment of allergic disease is based on:

• allergen avoidance;

• pharmacological management including disodium cromoglycate, theophyline, leukotriene antagonists, and local corticosteroids; and

• immunotherapy.

The treatment approaches using peptides, modified allergens, or allergens linked to TLR ligands such as CpG or Flagellin have the disadvantage that each allergen would have to go through clinical trials.

Although specific antagonists to other cytokines appear to be an attractive target for treatment, it is increasingly unlikely that they will be clinically successful in competition with anti-IgE, inhaled corticosteroids, and leukotriene antagonists.

Critical thinking: Severe anaphylactic shock (see p. 442 for explanations)

Sixty-two-year-old Mrs Young was stung by a bee from a hive in her back garden. Harvesting the honey had left her with several stings during the course of the summer. Several minutes after the recent sting she complained of an itching sensation in her hands, feet, and groin accompanied by cramping abdominal pain. Shortly afterwards she felt faint and acutely short of breath. Moments later she collapsed and lost consciousness. Her husband, a doctor, noticed that her breathing was rapid and wheezy and that she had swollen eyelids and lips. She was pale and had patchy erythema across her neck and arms.

On examination her apex beat could be felt, but her radial pulse was weak. Her husband immediately administered 0.5 mL of 1/1000 epinephrine (adrenaline) intramuscularly and 10 mg of chlorpheniramine (chlorphenamine) (an H₁-receptor antihistamine) intravenously with 100 mg of hydrocortisone. She regained consciousness and her respiratory rate dropped. By the following day she had recovered completely. Results of investigations at this time are shown in the table.

Investigation	Result (normal range)
hemoglobin (g/dL)	14.2 (11.5–16.0)
white cell count (× 10⁹/L)	7.5 (4.0–11.0)
neutrophils (× 10⁹/L)	4.4 (2.0–7.5)
eosinophils (× 10⁹/L)	0.40 (0.04–0.44)
total lymphocytes (× 10⁹/L)	2.4 (1.6–3.5)
platelet count (× 10⁹/L)	296 (150–400)
serum immunoglobulins IgG (g/L) IgM (g/L) IgA (g/L) IgE (IU/mL)	10.2 (5.4–16.1) 0.9 (0.5–1.9) 2.1 (0.8–2.8) 320 (3–150)
RAST bee venom wasp venom	class 4 class 0
skin prick tests	grade (0–5)
bee venom (10 μg/mL)	3+