Most allergens are proteins

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.

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:

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 half-life of IgE is short compared with that of other immunoglobulins

The concentration of IgE in the serum of normal individuals is very low compared to all the other immunoglobulin isotypes. Values range from <10–10 000 IU/mL, and the international unit (IU) is equivalent to 2.4 ng. Most sera contain <400 IU/mL (i.e. <1 μg/mL). The reasons why serum IgE is so low include:

• serum IgE has a much shorter half-life than other isotypes (~2 days compared with 21–23 days for IgG);

• IgE is produced in small quantities and is only produced in response to a select group of antigens (allergens and parasites); and

• IgE antibodies are sequestered on the high-affinity receptor on mast cells and basophils.

Q. What fundamental reason explains the low production of IgE?

A. The class switch from IgM to IgE happens infrequently and is controlled by T cells. The position of the IgE constant region gene is towards the distal end of the Ig gene stack (see Fig. 9.17), but the position alone cannot explain the infrequency of the switch to IgE.

The half-life of IgE in the serum has been measured both by injecting radiolabeled IgE and by infusing plasma from allergic patients into normal and immune-deficient patients.

The half-life of IgE in serum is less than 2 days; by contrast, IgE bound to mast cells in the skin has a half-life of approximately 10 days.

The low quantities of IgE in the serum must reflect a more rapid breakdown of IgE, as well as removal from the circulation by binding onto mast cells.

The most important site of breakdown of IgE is thought to be within endosomes where the low pH facilitates breakdown of free immunoglobulin by cathepsin.

Serum is constantly being taken up by endocytosis. Many macromolecules including IgE degrade in the endosome. One major exception is IgG, which is protected by binding to the neonatal Fc gamma receptor, FcγRn (Fig. 23.4).

Fig. 23.4 Endocytosis of plasma

Endocytosis of plasma contributes to the short half-life of IgE as plasma proteins are taken up and the pH falls because of lysosomes combining with the endosome. At low pH IgG including IgG4 molecules bind to the neonatal Fc γ receptor (FcγRn). By contrast, IgE molecules do not bind to FcγRn so are not protected and are digested by cathepsin. As the endosomes recirculate, the pH rises to 7.4 and the undamaged IgG molecules are released into the circulation. The FcγRn includes a molecule of β₂-microglobulin. In keeping with this model the half-life of IgG is shorter than normal in mice that have had the gene for β₂-microglobulin removed (or knocked out).

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.

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.

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:

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:

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: