Allergic symptoms are caused by an initial systemic histamine release by activated basophils and mast cells, that may lead to shock with laryngeal edema, lower-airway obstruction and hypotension. This is why basophils are considered with mast cells to be the key cells in allergic diseases.
Immunoglobulin E (IgE) is a class of antibody (or immunoglobulin "isotype") that has only been found in mammals. It plays an important role in allergy, and is especially associated with type 1 hypersensitivity. There are receptors (FcεR) for the constant region of IgE, the Fc region, on several types of cells, including Mast cells and Basophils. Basophils contain many granules inside the cell, which are filled with a variety of active substance triggering an allergic response upon degranulation. The cells are activated and start degranulation when the IgE antibody, bound to an allergen which can bind to the specific variable region of the IgE, the Fab region, bind to the Fc receptor
In most cases, a positive skin test is used in identification of allergies, but the activation of basophilic granulocytes with anti-IgE, the expression of the CD63 antigen on the cell surface (plasma membrane) allows identification of the allergen responsible for the hypersensitivity reaction without performing the common scratch test. Only a little amount of blood is needed for this experiment, which makes it comfortable to use since one can perform it in parallel to a normal blood checkup. It can be used for different allergies (e.g. bee venom, drugs, contrast media [1] ).
Degranulated cell expose CD63 [2] molecules on their outer cell membrane, hence the granules, which contain CD63 molecules on their inner surface, merged with the cell membrane. The inner cell surface of the granules becomes the outer cell surface of the basophil /mast cell during degranulation process.
| materials | purpose |
|---|---|
| BSB | basophil stimulation |
| IL-3 | basophil stimulation |
| allergen | |
| EDTA | degranulation stopper |
| marker | marks the basophils |
| lyse solution | lyses red blood cells |
| centrifuge | spin down red blood cells |
| PBS | washing away lyse solution |
| FACS | counting cells |
As flow cytometry is a valuable tool for analyzing large numbers of cells and for identifying cell populations, even at low concentrations, the percentage of basophils activated after in vitro stimulation by allergens and expressing the CD63 marker can be determined. The CD63 marker is a fluorescein isothiocyanate labeled antigen which can bind to an CD63 protein and is used to sort the cells via FACS(Fluorescence activated cell sorting/sorter). This FITC labeled antigen emits light at a wavelength of 530 nm. As the emitted fluorescence intensity is proportional to the binding sites of each single cell, the intensity will increase according to the number of FITC- conjugated antibodies bound to CD63 expressing cells.
A test tube is prepared with basophil stimulation buffer (BSB) including Interleukin 3 and an allergen which is to be tested. The blood sample is added and the tube is incubated at 37 °C for several minutes, to ensure that the allergens can bind to the IgE. By adding EDTA to the test tube, the degranulation process is stopped immediately. After degranulation a CD63 marker (labeled antibodies) is added to the test tube. Several minutes at room temperature gives the marker time to bind to the CD63 proteins on the cell membrane of the basophil. A lysing step is performed to lyse the red blood cells. Because they outnumber by far the leucocytes they need to be removed to do a FACS analysis of the basophils.
Allergies, also known as allergic diseases, refer to a number of conditions caused by the hypersensitivity of the immune system to typically harmless substances in the environment. These diseases include hay fever, food allergies, atopic dermatitis, allergic asthma, and anaphylaxis. Symptoms may include red eyes, an itchy rash, sneezing, coughing, a runny nose, shortness of breath, or swelling. Note: food intolerances and food poisoning are separate conditions.
A mast cell is a resident cell of connective tissue that contains many granules rich in histamine and heparin. Specifically, it is a type of granulocyte derived from the myeloid stem cell that is a part of the immune and neuroimmune systems. Mast cells were discovered by Paul Ehrlich in 1877. Although best known for their role in allergy and anaphylaxis, mast cells play an important protective role as well, being intimately involved in wound healing, angiogenesis, immune tolerance, defense against pathogens, and vascular permeability in brain tumours.
Hypersensitivity refers to undesirable reactions produced by the normal immune system, including allergies and autoimmunity. They are usually referred to as an over-reaction of the immune system and these reactions may be damaging and uncomfortable. This is an immunologic term and is not to be confused with the psychiatric term of being hypersensitive which implies to an individual who may be overly sensitive to physical and/or emotional stimuli. Hypersensitivity reactions can be classified into four types.
Basophils are a type of white blood cell. Basophils are the least common type of granulocyte, representing about 0.5% to 1% of circulating white blood cells. However, they are the largest type of granulocyte. They are responsible for inflammatory reactions during immune response, as well as in the formation of acute and chronic allergic diseases, including anaphylaxis, asthma, atopic dermatitis and hay fever. They also produce compounds that coordinate immune responses, including histamine and serotonin that induce inflammation, heparin that prevents blood clotting, although there are less than that found in mast cell granules. Mast cells were once thought to be basophils that migrated from blood into their resident tissues, but they are now known to be different types of cells.
Immunoglobulin G is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG antibody has two paratopes.
Immunoglobulin D (IgD) is an antibody isotype that makes up about 1% of proteins in the plasma membranes of immature B-lymphocytes where it is usually co-expressed with another cell surface antibody called IgM. IgD is also produced in a secreted form that is found in very small amounts in blood serum, representing 0.25% of immunoglobulins in serum. The relative molecular mass and half-life of secreted IgD is 185 kDa and 2.8 days, respectively. Secreted IgD is produced as a monomeric antibody with two heavy chains of the delta (δ) class, and two Ig light chains.
Immunoglobulin E (IgE) is a type of antibody that has been found only in mammals. IgE is synthesised by plasma cells. Monomers of IgE consist of two heavy chains and two light chains, with the ε chain containing four Ig-like constant domains (Cε1–Cε4). IgE is thought to be an important part of the immune response against infection by certain parasitic worms, including Schistosoma mansoni, Trichinella spiralis, and Fasciola hepatica. IgE is also utilized during immune defense against certain protozoan parasites such as Plasmodium falciparum. IgE may have evolved as a defense to protect against venoms.
Type I hypersensitivity, in the Gell and Coombs classification of allergic reactions, is an allergic reaction provoked by re-exposure to a specific type of antigen referred to as an allergen. Type I is distinct from type II, type III and type IV hypersensitivities. The relevance of the Gell and Coombs classification of allergic reactions has been questioned in the modern-day understanding of allergy, and it has limited utility in clinical practice.
Omalizumab, sold under the brand name Xolair, is a medication used to treat asthma, nasal polyps, and urticaria (hives).
Allergic conjunctivitis (AC) is inflammation of the conjunctiva due to allergy. Although allergens differ among patients, the most common cause is hay fever. Symptoms consist of redness, edema (swelling) of the conjunctiva, itching, and increased lacrimation. If this is combined with rhinitis, the condition is termed allergic rhinoconjunctivitis (ARC).
A patch test is a diagnostic method used to determine which specific substances cause allergic inflammation of a patient's skin.
In immunology, a Fc receptor is a protein found on the surface of certain cells – including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells – that contribute to the protective functions of the immune system. Its name is derived from its binding specificity for a part of an antibody known as the Fc region. Fc receptors bind to antibodies that are attached to infected cells or invading pathogens. Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or infected cells by antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. Some viruses such as flaviviruses use Fc receptors to help them infect cells, by a mechanism known as antibody-dependent enhancement of infection.
The high-affinity IgE receptor, also known as FcεRI, or Fc epsilon RI, is the high-affinity receptor for the Fc region of immunoglobulin E (IgE), an antibody isotype involved in the allergy disorder and parasites immunity. FcεRI is a tetrameric receptor complex that binds Fc portion of the ε heavy chain of IgE. It consists of one alpha, one beta, and two gamma chains connected by two disulfide bridges on mast cells and basophils. It lacks the beta subunit on other cells. It is constitutively expressed on mast cells and basophils and is inducible in eosinophils.
Allergic inflammation is an important pathophysiological feature of several disabilities or medical conditions including allergic asthma, atopic dermatitis, allergic rhinitis and several ocular allergic diseases. Allergic reactions may generally be divided into two components; the early phase reaction, and the late phase reaction. While the contribution to the development of symptoms from each of the phases varies greatly between diseases, both are usually present and provide us a framework for understanding allergic disease.
Talizumab (TNX-901) is a humanized monoclonal antibody that was under development by Tanox in Houston, Texas as a new-concept therapeutic for allergic diseases. The unique anti-IgE antibody was designed to target immunoglobulin E (IgE) and IgE-expressing B lymphocytes specifically, without binding to IgE already bound by the high affinity IgE receptors on mast cells and basophils. Talizumab was tested in clinical trials at National Jewish Medical and Research Center and other medical centers and allergy clinics across the U. S. and shown to be able to prevent allergic reactions to accidental exposure to peanuts, which is contained in many kinds of foods.
Degranulation is a cellular process that releases antimicrobial cytotoxic or other molecules from secretory vesicles called granules found inside some cells. It is used by several different cells involved in the immune system, including granulocytes and mast cells. It is also used by certain lymphocytes such as natural killer (NK) cells and cytotoxic T cells, whose main purpose is to destroy invading microorganisms.
CD16, also known as FcγRIII, is a cluster of differentiation molecule found on the surface of natural killer cells, neutrophils, monocytes, macrophages, and certain T cells. CD16 has been identified as Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b), which participate in signal transduction. The most well-researched membrane receptor implicated in triggering lysis by NK cells, CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). It can be used to isolate populations of specific immune cells through fluorescent-activated cell sorting (FACS) or magnetic-activated cell sorting, using antibodies directed towards CD16.
A drug allergy is an allergy to a drug, most commonly a medication, and is a form of adverse drug reaction. Medical attention should be sought immediately if an allergic reaction is suspected.
Type II hypersensitivity, in the Gell and Coombs classification of allergic reactions, is an antibody mediated process in which IgG and IgM antibodies are directed against antigens on cells or extracellular material. This subsequently leads to cell lysis, tissue damage or loss of function through mechanisms such as
The following outline is provided as an overview of and topical guide to immunology: