Allergic symptoms are caused by an initial systemic
histamine
Histamine is an organic nitrogenous compound involved in local immune responses, as well as regulating physiological functions in the gut and acting as a neurotransmitter for the brain, spinal cord, and uterus. Since histamine was discovered in ...
release by activated
basophil
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 inflammator ...
s and
mast cell
A mast cell (also known as a mastocyte or a labrocyte) 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 par ...
s, that may lead to shock with
laryngeal edema
Edema, also spelled oedema, and also known as fluid retention, dropsy, hydropsy and swelling, is the build-up of fluid in the body's tissue. Most commonly, the legs or arms are affected. Symptoms may include skin which feels tight, the area ma ...
, lower-airway obstruction and
hypotension
Hypotension is low blood pressure. Blood pressure is the force of blood pushing against the walls of the arteries as the heart pumps out blood. Blood pressure is indicated by two numbers, the systolic blood pressure (the top number) and the dias ...
. This is why basophils are considered with mast cells to be the key cells in allergic diseases.
Activation process
Immunoglobulin E (
IgE
Immunoglobulin E (IgE) is a type of antibody (or immunoglobulin (Ig) " isotype") that has been found only in mammals. IgE is synthesised by plasma cells. Monomers of IgE consist of two heavy chains (ε chain) and two light chains, with the ε c ...
) 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
Hypersensitivity (also called hypersensitivity reaction or intolerance) 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 s ...
. There are receptors (FcεR) for the constant region of IgE, the
Fc region
The fragment crystallizable region (Fc region) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune s ...
, 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
Fab or FAB may refer to:
Commerce
* Fab (brand), a frozen confectionery
* Fab (website), an e-commerce design web site
* The FAB Awards, a food and beverage award
* FAB Link, a European electricity link
* Flavoured alcoholic beverage or alcopop ...
, bind to the
Fc receptor
''In vitro'' allergy test method
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
Apitoxin or bee venom is the venom produced by the honey bee. It is a cytotoxic and hemotoxic bitter colorless liquid containing proteins, which may produce local inflammation. It may have similarities to sea nettle toxin.
Components
Bee venom is ...
,
drugs
A drug is any chemical substance that causes a change in an organism's physiology or psychology when consumed. Drugs are typically distinguished from food and substances that provide nutritional support. Consumption of drugs can be via inhalat ...
,
contrast media
A contrast agent (or contrast medium) is a substance used to increase the contrast of structures or fluids within the body in medical imaging. Contrast agents absorb or alter external electromagnetism or ultrasound, which is different from radio ...
[Böhm I et al. ''Pilot study on basophil activation induced by contrast medium''. Fundam Clin Pharmacol 2011;25:267 - 276]).
Degranulation
Degranulated cell expose
CD63
CD63 antigen is a protein that, in humans, is encoded by the ''CD63'' gene. CD63 is mainly associated with membranes of intracellular vesicles, although cell surface expression may be induced.
Function
The protein encoded by this gene is a memb ...
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.
Labeling and sorting
As
flow cytometry
Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles.
In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...
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
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging applications including flow cytometry. First described in 1942, FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group ...
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.
Procedure
A test tube is prepared with basophil stimulation buffer (BSB) including
Interleukin 3
Interleukin 3 (IL-3) is a protein that in humans is encoded by the ''IL3'' gene localized on chromosome 5q31.1. Sometimes also called colony-stimulating factor, multi-CSF, mast cell growth factor, MULTI-CSF, MCGF; MGC79398, MGC79399: the protein ...
and an
allergen
An allergen is a type of antigen that produces an abnormally vigorous immune response in which the immune system fights off a perceived threat that would otherwise be harmless to the body. Such reactions are called allergies.
In technical terms ...
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
Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2sub>2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes eve ...
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
White blood cells, also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from mult ...
they need to be removed to do a FACS analysis of the basophils.
See also
*
Innate immune system
The innate, or nonspecific, immune system is one of the two main immunity strategies (the other being the adaptive immune system) in vertebrates. The innate immune system is an older evolutionary defense strategy, relatively speaking, and is the ...
*
Allergy
Allergies, also known as allergic diseases, refer 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 derma ...
*
B cell
B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system. B cells produce antibody molecules which may be either secreted or ...
*
Fc receptor
References
{{Reflist
External links
CMA clinical molecular allergy
Cell biology
Granulocytes
Human cells