Role Of Basophils In Allergic Sensitization In Humans Biology Essay


The prevalence of allergic disorders is increasing in the developed world and includes atopic asthma, several food allergies, atopic dermatitis, allergic rhinitis and anaphylaxis. Anaphylaxis is a potentially fatal systemic allergic response, which develops within seconds or minutes after contact with allergens (1, 2). It is important to understand that this is a severe allergic reaction that rapidly takes place after exposure to non-infectious environmental substances (3). Allergic diseases, such as anaphylaxis, are related to the expansion of allergen-specific T helper 2 (Th2) cells and involve allergen-specific IgE. They are both reactive to environmental matters that are normally not dangerous (1).

After contact with a specific antigen in sensitized subjects, an allergic inflammation is produced. Sensitization means that an allergen is capable to induce a Th2-cell reaction, in which IL-4 drives IgE production (1, 2, 4). The sensitization starts with an allergen that can be sampled by antigen presenting cells like dendritic cells. The activated dendritic cells mature and travel to regional lymph nodes, where they present peptides obtained from the processed antigen in the context of major histocompatibility complex II molecules to naïve T cells (1, 5, 6). The induction of adaptive cellular immunity needs also a second signal of co-stimulatory molecules and a third signal of instructive cytokines like IL-4 (1, 5). In the presence of these three signals, naïve T cells become T helper 2 cells that produce IL-4, IL-5 and IL-13. With these cytokines and the appropriate co-stimulatory molecules, B cells undergo immunoglobulin class-switch recombination. This process consists of the rearrangement of the gene segments that predetermine the immunoglobulin heavy chain in such a way that antibody of the IgE class is produced (1, 2, 7). IgE is diffused locally, enters the lymphatic vessels, consequently enters the blood and circulates then systemically. Once in the interstitial fluid, the IgE binds to the high affinity receptor FcεRI on mast cells and basophils that are present in the tissue. In this way, mast cells and basophils become sensitized to respond when the host is exposed to the allergen for the second time (1).

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After sensitization, a single allergen exposure produces an acute reaction, called the early-phase reaction. Mostly, this is followed by a late-phase reaction and if the antigen is consistently exposed, chronic inflammation develops (1). The early-phase reaction can occur within minutes after exposure to an antigen, and is an IgE-mediated hypersensitivity reaction. The reactions can be localized, but in the case of anaphylaxis the reaction is systemic. The response starts with IgE that binds to the FcεRI receptor on basophils and mast cells and is cross linked by allergen. This result in the release of a wide variety preformed and newly produced mediators, which lead to vasodilatation, oedema because of vascular permeability, and in the affected organs it leads to acute functional differences. Several released mediators contribute the development of the late-phase responses, by supporting the local recruitment and activation of Th2 cells and some other cells (1).

As mentioned before, the initiation of the adaptive cellular immune system needs three signals of the antigen presenting cell. The signals include the peptide-MHC presentation, co-stimulatory molecules and instructive cytokines (1, 5, 6). They are responsible for the generation of different subsets of T helper cells to manage different kinds of defensive immunity (5). The signals that are involved with the Th1 and Th17 cell differentiation by dendritic cells are well known, but the mechanisms that induce the Th2 cell differentiation in vivo are not that well understood (5, 6, 8). It is quite clear that the dendritic cells are the main players in the initiation and development of most effector and regulatory T cell classes. But the relative input of active dendritic cells in the development of Th2 effector reactions is less clear (6). Despite the overwhelming proof that IL-4 is required for most of the Th2 reactions, it has never been demonstrated that the dendritic cells produce IL-4 (5, 6, 8). For that reason, it was thought that the Th2 reactions would happen by default, in the absence of Th1 of Th17 instructive cytokines (5, 8). From another vision, IL-4 is produced by an additional innate immune cell type like basophils, which make IL-4 available for the activation of the Th2-differentiation (4-6, 8).

In summary, the dendritic cells are best known as antigen presenting cells that induce adaptive immune responses, likely in the presence of IL-4 producing basophils (4-6, 8). However, three papers showed that dendritic cells are not required for the initiation of Th2 reactions (4, 6, 8, 9). They suggest that basophils act as antigen presenting cells and thereby initiate the Th2 reactions to protease allergens (8), helminthic parasites (9), or antigen-IgE complexes in vivo (4). The MHC class II-positive interleukin-4-producing basophils are both essential and sufficient for the triggering of Th2 differentiation (6). On the other hand, a study of Hammad and others, also tested the role of basophils in a model of house dust mite-drive asthma (5). They demonstrated that dendritic cells were essential and sufficient for the triggering of Th2 immunity and characteristics of asthma, but basophils were not required (5). Hammad et al believe in a model in which the basophils amplify but the DCs induce Th2 immunity to HDM antigen (5).

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What also is very important to notice, Hammad and colleagues pointed out that the conclusions on the relative in vivo involvement of dendritic cells versus basophils as antigen presenting cells and inducers of the Th2 differentiation, are different from model to model (5). These are not only controlled by the route of injection and the sort of allergen, but could also be dependent on the approach in which cells are depleted and refined for functional experiments (5). This corresponds with a statement of Thomas Wynn, who says that it is necessary to find out whether all antigen-specific Th2 reactions are initiated by the basophil-dependent mechanism as suggested in the three papers he discussed (4, 6, 8, 9). Maybe specific DC subsets or other antigen presenting cell populations outdo basophils in other situations (6).

Besides these criteria, most of the research of the involvement of the basophil in the Th2 differentiation has been done in vitro and in vivo with animals. It is unclear how the conclusions of these experiments are relevant to humans. Do the basophils play only a role in the Th2 effector immunity, are they accessory cells for the DCs or are they the main player in the induction of Th2 immune responses? This leads to the following research question: What is the role of basophils in allergic sensitization in humans?


It is important to better understand the mechanisms that are responsible for many allergic diseases. If the mechanisms are clear, potential novel therapies can be tested (10). Much of the research efforts have lead to the development and analysis of models in animals. Such approaches have remarkable advantages, but the results received with laboratory animals are not automatically relevant to human subjects (10).

For that reason it is important to determine the role of basophils in the allergic sensitization in humans. If basophils play an important role in the induction of Th2 responses, new therapies can be tested and maybe in the future used in practice.

Research plan

To test whether the basophils have a role in de sensitization in atopic patients, we would like to do three different experiments. We hope to find some consistent variations between the basophils of atopic and healthy persons. Maybe these differences can be related to the way the basophils act like APCs and as a result contribute to the way allergic sensitization develops.

Experiment 1 - ex vivo characterization of basophils: Genome-wide expression array

This experiment focuses on the differences in the genome expression profile between basophils of atopic patients versus healthy controls. It is a kind of characterization without hypotheses; are there any consistent variations possible between the expression profiles of these groups? And do these variations differ when the basophils are activated?

For this experiment we would like to collect heparin blood samples of atopic patients and healthy controls. By using the magnetic-activated cell sorting (MACS)-technology we will sort out the basophils from the blood samples. We will look at the basophils in a basal state and after stimulation with IgE-cross linking. With a genome-wide expression array we will monitor the RNA products of many genes at once. By using this technique we can identify clusters of genes that specific come to expression in either the atopic subjects or the healthy controls.

It is possible that the expression profiles are different from the atopic patients and the healthy patients, for example in the atopic patients are more genes expressed which are related to the mechanisms of Th2 differentiation. Hopefully this gene expression profiling will help us to find a hypothesis for further experiments.

Experiment 2 - ex vivo characterization of basophils: directed to measurable differences

This experiment focuses on the actually measurable differences between basophils of atopic patients and healthy controls. After collecting heparin blood and cell sorting with the MACS-technology, we would like to look at the basophils with a basophil activation test in a basal state and after activation with IgE-cross linking. Besides the basophil activation test, we would like to look at the up-regulation of specific surface markers, such as co-stimulatory molecules and CD40 ligand, and the production of cytokines like IL-4. The specific surface markers will be labeled with fluorescent antibodies and the labeled cells can be separated from the unlabeled cells in an electronic fluorescence-activated cell sorter (FACS) (11). The production of cytokines like IL-4 will be measured in the supernatant by an ELISA test. The basophil activation test, determination of up-regulation of surface markers and production of cytokines, can hopefully give us some interesting results: are there any differences in activation of the basophils at all? And do these differences contribute to the understanding of allergic sensitization in atopic patients?

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Experiment 3 - sensitization differences in Th2 differentiation

In this experiment we would like to look at the effects of depletion of basophils or monocytes (dendritic cells) in the process of sensitization. After collecting total blood cells from heparin blood samples of allergic and healthy subjects, either the basophils or the monocytes are sorted out with the MACS-technology. The experiments will be done in the absence or presence of a new allergen, an allergen that the atopic individuals has not been exposed to as detected by the absence of specific IgE. After these processes of cell sorting and activation, we look at the Th2 differentiation of the T cells. This will be done by determination of cytokines in the supernatant with an ELISA or with an intracellular FACS staining of the cytokines (such as IL-4, IL-5 and IL-10) in the T cells. More specific, with the ELISA we measure the total amount of produced IL-4 and with the FACS we measure the total amount of cells that produce IL-4.

By testing the abilities of dendritic cells and basophils to act as APCs, we can determine which cells are the main players in the allergic sensitization. Perhaps the relative involvement of DCs and basophils in Th2 differentiation is different in atopic patients, which lead to the allergic sensitization.

Methods, techniques

Blood sampling

Heparin blood will be sampled from atopic and normal subjects and total blood cells will be collected.

Magnetic-activated cell sorting (MACS)

With the MACS-technology you can label the cells of interest, whereby MACS-conjugated antibodies directed against cell-specific surface antigens are used (12). These MACS-conjugated antibodies are antibodies which are labeled with a magnetic particle. When a tube with labeled cells is located in a magnetic field, the cells will separate according to their magnetic label (12).

IgE cross linking

We will activate the basophils with IgE- specific antibodies. By using this anti-IgE, all the IgE will be cross linked. Culturing the basophils in medium will take about 24 hours in an incubator.

Genome wide array

With the genome-wide array, also genome-wide expression analysis or microarray, we will determine if there are any consistent differences in gene expression of atopic patients as compared to healthy controls. After isolating mRNA from the basophils, the samples will be converted to cDNA by reverse transcription using fluorescently labeled nucleotides. The cDNA mixtures will be applied to a microarray and the cDNA hybridizes with any complementary DNA on the microarray. Ideally, these DNA fragments represent all the genes of an organism. Following incubation, the microarray will be washed and the fluorescence scanned. The fluorescence intensity at each spot is a measure of the expression of the gene represented by that spot in the sample (13).

Basophil activation test

The basophil activation test will be done by a flow cytometry. Normally the first step of these assays is identification of basophils by specific fluorescent antibodies such as anti-IgE, but in our experiment the basophils are already sorted out by the MACS-technology. The second step is monitoring basophil activation (with or without allergen challenge) by detecting surface expression of degranulation markers such as CD69 or CD203c (14, 15).


The specific surface markers on basophils will be labeled with fluorescent antibodies and the labeled cells can be separated from the unlabeled cells in an electronic fluorescence-activated cell sorter (FACS) (11). In the FACS individual cells travelling single line in a fine stream pass through a laser beam and are monitored for fluorescence (11).


With the enzyme-linked immune sorbent assay (ELISA) we can detect and assay specific molecules, like the total amount of produced cytokine IL-4. The sensitivity of antibodies that recognize a specific target molecule is enhanced by the use of enzyme labeled secondary antibodies. For example, the enzyme may produce some chemicals that lead to the production of local colored residues. This reveals the location of the secondary bound antibody and hence the position of the antibody-specific target molecule complex. This enzyme amplification makes enzyme-linked methods very sensitive (11).

Intracellular FACS staining

Several advances in FACS methodology have contributed to the expanded use of FACS instruments, for example the development of new methods for intracellular molecules (16). In this way, the intracellular cytokines of T-cells will be labeled with fluorescent antibodies. The principle of cell sorting is the same as the FACS.


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