Type - 1 Hypersensitivity Reaction

Rosina Wallis Biomedical Sciences year 2 23rd March 2005

Sensitised Guinea Pig Ileum. (Hypersensitivity type1)

Aims.

An experiment was conducted to investigate hypersensitivity type 1 (allergic reactions) and the effects that different doses (0.2 10-4 m and 0.4 10-4 M) of histamine had on the guinea pig ileum (tissue). The different effects that mepyramine and carbachol had on the guinea pig ileum were also investigated.

A piece of tissue from a guinea pig’s ileum, which had been 1, non-sensitised and 2, sensitised to egg albumin were exposed to mepyramine, carbachol and different doses of histamine. The experiment was to illustrate any differences that occurred between the sensitised guinea pig ileum and the non-sensitised guinea pig ileum. The control experiment was the non-sensitised guinea pig’s ileum (tissue).

Introduction.

The immune/ inflammatory system is designed to fight against infections foreign substances/molecules that invade the body, these foreign substances can be specifically recognised by the white cells in the blood stream. T-Lymphocytes pass through the bloodstream, lymph nodes and the connective tissues searching out foreign substances i.e. antigens, which are located on pathogens and abnormal cells.

The immune system is divided into two classes, the innate system (natural immunity) and the innate system, which is not capable of specifically recognising antigens, and resistance to infection is not determined by a returning infection, given that it does not stimulate the memory cells. However basophils and macrophages (antigen presenting cells) do play an important role with respect to effector cells in cell-mediated immunity. T-cells recognise protein antigens (foreign molecules), which are presented to them by the macrophages.

Antigens are foreign molecules found on viruses, bacteria and fungi, pollen and toxins and can induce an immune/allergic response, i.e. allergies.

“Allergens are usually specific protein antigens (pep-tides) that stimulate immune hypersensitivity by reacting with IgE or IgG antibodies and T-cells”. Current Allergy & Clinical Immunology, March 2004 Vol 17, No. 1

The other class of immunity is known as the adaptive immunity (antibody mediated immunity), which is mediated by B-lymphocytes and is differentiated into active and passive immunity. The adaptive immunity is highly specific because on the first exposure to an antigen it produces memory B and T-lymphocytes consequently it can make a distinction between different viruses. Therefore the immune/ inflammatory resistance is improved by a second bout of infection (Clonal selection theory), due to the memory cells, which are derived from the B and T-lymphocytes and is known in humoral immunity. Additionally it is the TH lymphocytes, which influence mast cells.

“The T-cell system is involved, because IgE production is strongly if not absolutely dependent on T-cell help”. IgG4 antibody assays in allergy diagnosis .R. C. Aalberse, J. Schuurman, R. van Ree and S. Stapel

An antibody molecule is a Y shaped protein that comprise of 2 identical heavy and 2 identical light chains (four polypeptide chains). Each has a specific function and they have 2 FAB fragments that hold the antibody activity, where the antigen binds, and 1 FC (fragment crystal) portion; where the Immunoglobulin E (IgE) binds. In hypersensitivity the antigen binding site is exposed, enabling the binding of a multi-valent antigen that cross links on the receptors this in turn triggers a response in the releasing of mediators such as histamine. Antibodies recognise and respond to antigens and an antigen, which stimulates the synthesis of IgE, is known as an allergen and is involved in a hypersensitivity response, also haptens or incomplete antigens are capable of triggering off type 1 immediate allergic reaction.

Rearrangements in the DNA creates various antibody molecules known as immunoglobulins, the body produces 5 classes, IgM, IgD IgA, IgG, and igE. These immunoglobulins travel through the bloodstream and merge with antigens thus, deactivating antigens this is achieved because when an antigen is present the dendritic cells are altered.

T-lymphocytes; produce antibodies and cytokines to the innate immunity, macrophages (antigen-presenting cells), present antigens to the helper T-cells, (B-cells, are also antigen presenting cells). Synthetic compounds, toxins, and their native toxins are capable of affecting an immune or inflammatory response, which is achieved through the T.H Lymphocytes (helper T.cells). These helper cells are derived from virgin/naive T4 and T8 –lymphocytes and are triggered through the recognition of cell-associated antigens, which have several subtypes including helper T cells T4 (regulatory cells), or cytotoxic T cells CD4, which destroy virally infected cells and tumour cells and suppressor T-cells.

The TH lymphocytes influence mast cells (in the endothelial lining in connective tissue), and are capable of acting as sensory transducers e.g. contractile responses follows an exposure to a specific antigen. An Immunoglobulin E (IgE), which is an antibody, migrates to the mask cells, which are made in the bone marrow.

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Immunoglobulins E (IgE) are produced by the plasma cells and they possess a high affinity to bind to FC receptors sites on the mast cells. However this only occurs if a person has had at least one preceding exposure to the allergen. The mask cells contain and release many biologically active secretory substances including histamine, which in turn bind onto the histamine 1 (H1) receptor on muscle (cells) surface.

“ The binding of allergens to IgE on mast cells triggers the release of several mediators, of which histamine is the most prevalent”. J Clin Invest, December 2001, Volume 108, Number 12, 1865-1873.

Guinea pig ileum is made up of smooth and longitude muscles and contains different receptors located on the cells of the muscle surface. The experiment was to investigate the nature of how egg albumin (allergen/antigen) and by what means histamine, mepyramine and carbachol elicits a response, or attempt to block a response to the guinea pig ileum (tissue) by binding onto the same or similar receptors. Before this investigation the sensitised guinea pig ileum was treated with a sensitising dose of egg albumin and two weeks later another dose of egg albumin was given. The second guinea pig ileum (the control) was given a dose of a normal saline solution.

It is suggested from this experiment, that when histamine binds onto H1 receptors located on tissue (guinea pig ileum) it causes the smooth muscle/ tissue to contract, via a primary response to vasodilation and an increase in the permeability of the capillary vessels, which are located in the endothelial lining of all smooth muscle. Consequently, the permeability and vasodilation of the blood vessels enables more antibodies, in the bloodstream to travel to the region of the antigens, (thus aiding a damaged tissue).

Histamine is a natural agonist acts on H1 and H2 receptor thus produces a reaction also a mediator in hypersensitivity and when a non-sensitised tissue is exposed to histamine, histamine acts by binding onto the H1 receptors this action is associated with an allergic reaction also histamine primarily acts on smooth muscle (thus contraction in smooth muscle) and blood vessels, which causes vasodilation and an increase in permeability. Egg albumin like histamine works on the H1 receptors, which are located on the cells of longitude and smooth muscle. There are three different histamine receptors extensively located throughout mammalian tissue these are H1 H2 (stimulates gastric secretion and stimulates the heart rate) and H3 and they all elicit a response in many different ways when different substances are introduced to the body. Additionally, H1 antagonist drugs prevent the effects of an agonist, and many H1 receptor antagonists are prescribed clinical drugs used to alleviate symptoms that are involved in allergic reactions (i.e. hay fever watery eyes). The binding of IgE to the FC receptor on the mask cells surface can cause an anaphylactic response.

The binding to H1 receptors is thought to mediate histamine-induced symptoms in an allergen response.

A suggestion was made, that egg albumin stimulates the release of histamine in the mast cells. When the egg albumin is first introduced to the sensitised guinea pig ileum tissue it illustrates a response that is similar to that of histamine, although there is a difference in that the response is believed to occur after the IgE antibody binds to specific FC receptors on the mast cells surface, which acquire the ability to act as sensory inducers.

Consequently when the histamine is released and binds onto the H1 receptors, this blockade immediately results in a bi-phasic response of vasoconstriction, which is instantly followed by peripheral vessel dilation, resulting in a contractile response of the smooth muscle in the guinea pig ileum. This is believed to be an immediate hypersensitivity response, where the body overproduces IgE antibodies in response to the over release of histamine, basically known as an over active immune response.

Mepyramine, which is an antagonist which like histamine, also binds onto the H1 receptor this in turn, blocks histamine from binding onto the H1 receptors that are located on the surface of the ileum. Suffice to say that, when mepyramine binds onto the H1 receptors the histamine cannot bind on, once the binding onto a receptor on the surface of the (guinea pig ileum) tissue occurs, no other substance can then bind onto the same receptor (a similarity to the lock and key theory, enzymes).

Carbachol acts on acetylcholine receptors, and it is suggested that carbocherol was used as an experimental implement, to demonstrate that the tissue in the guinea pig ileum was still alive. Carbachol acts on both the muscarinic receptors and acetylcholine receptors on the muscle. Neuroeffector junctions have different subtype receptors and muscarinic receptor is one of them. The acetylcholine stimulates a contractile response by releasing acetylcholine that acts on the choline receptors, located at the cholinergic site on longitude muscle cells on the guinea pig ileum. Contraction of intestinal smooth muscle can occur via several neurotransmitters acting directly on the muscle cells. Therefore it is suggested that this is what caused the muscle to contract.

“In vitro the muscarinic receptors mediating the contraction of the detrusor muscle in Cynomolgus monkeys and guinea pigs using carbachol as the agonist.” Lai FM, Cobuzzi A, Spinelli W

Results.

The results that were recorded here are of a qualitative data, please see diagrams below text.

Different dilutions of histamine were used in the first experiment this was to see which histamine would give the greatest response. Because the two responses were different from each other and it was decided that the 0.2 ml and 0.4 ml of histamine at 10-4 M was the most effective therefore this was the dilution that was used in the entire experiment. Also carbachol and mepyramine was a 10-4 molar.

Non-sensitised tissue

Control, when the 0.4 and 0.2 ml of 10-4 M histamine was added there was a contractile response in the guinea pig ileum. However the oscillograph measured a higher contractile response when 0.4 10-4 M of histamine was used opposed to the 0.2 10-4 M of histamine.

There was no response when on both occasions that the 1ml of egg albumin was added to the guinea pig ileum tissue. Seeing that this tissue was not sensitised to egg albumin this result would be correct because no mask cells were available to release histamine, i.e. antibodies were not available to elicit an immune response.

When the 0.2 ml of 10-4 mepyramine was added a contractile response was not seen possibly because mepyramine is an antagonist that also binds onto h1 receptors and the receptors already had the histamine bound onto them, thus blocking a response to mepyramine. When the 0.4 ml of 10-4 M of carbachol (with no wash in-between time) was added a contractile response was seen and recorded. Carbachol was possibly used to demonstrate that the tissue was still alive.

The oscillograph measured sensitised tissue 1 and when 0.2 and 0.4 ml of 10-4 M of histamine was added a contractile response, was noted and this was recorded.

When 1 ml of egg albumin was added the first time there was a contractile response. However, the second time 1ml of egg albumin was added a contractile response was not noted. Without washing 0.4 10-4 M of histamine was added a similar contractile response to when the egg albumin was first added was noted and recorded.

When 0.2 10-4 M of mepyramine was added followed by 0.4 ml of 10-4 histamine a contractile response was not noted and this was recorded.

When 0.4 ml of 10-4 carbachol was added a contractile response was noted and recorded. Again this suggests that the tissue was still alive because carbachol works on the acetylcholine receptors.

Sensitised tissue 2, a contractile response was noted and recorded for 0.2 and 0.4ml of 10-4 M histamine. The contraction was slightly higher when the 0.4ml (larger amount) of histamine was added. When the 1ml of egg albumin was added the first time a contractile response was noted. However when the second dose was added a contractile response was not noticed

When 0.2 ml of 10-4 mepyramine and 0.4 ml of 10-4 histamine was added 30 seconds later a contractile response was not noted this too was recorded again (possibly because h1 receptor histamine bound).

0.4 ml of 10-4 carbachol was added and a contractile response was noted and recorded, this possibly illustrated that the tissue was still alive.

Discussion

There is much evidence and data made available via scientific papers in journals, which, indicates that allergens are specific antigens, which are capable of eliciting an immune response, by causing the mast cells to release biological active molecules such as histamine. Additionally the production of antigen/allergen specific IgE cannot occur unless the body firstly has been exposed to the said antigen (i.e. egg albumin).

However after conducting the control experiment it was determined that on the Non-sensitised guinea pig ileum and on the first exposure to the (EA) egg albumin contractile response was not noticed this could have been because the control (guinea pig ileum) tissue had not been sensitised to egg albumin beforehand. Also conducting the experiment on the Non-sensitised guinea pig ileum (control) 0.2 and 0.4 of 10-4M histamine was added contractile responses of the tissue was noted, however when the 0.4 of 10-4 M was added a greater contraction was measured, than the 0.2ml of 10-4 M of histamine. This response was a naturally elicited response via the histamine binding onto the h1 receptors on the surface of the tissue.

This possibly illustrated the evidence that histamine is a mediator that created a contractile response as part of an allergic reaction; the seen contractions were possibly mediated by the binding of histamine to the H1 receptor. Additionally, it seemed evident that the higher the concentration of histamine, the stronger the tissue (guinea pig ileum) contracted, the histamine possibly was not only bound to the H1 receptors but quite likely bound to the H2 receptors as well. It seemed evident that the shot of histamine from outside the guinea pig ileum also creates a contractile response that occurs via histamine binding on the H1 receptors. This blockade immediately results in a bi-phasic response of vasoconstriction and is immediately followed by peripheral vessel dilation, thus a contractile response. Also it can be determined that this was an innate immune response.

When 0.2ml of 10-4 M mepyramine followed by 0.4 ml of 10-4 histamine was added to the Non-sensitised (guinea pig ileum) tissue, the tissue did not contract a reason for this was because mepyramine which is an antagonist and was bound onto H 1 receptors, on the surface of the ileum this in turn disabled the histamine from binding onto the H1 receptor. (Similar to the lock and key theory, enzymes).

Sensitised 1 guinea pig ileum on the first exposure to 1 ml of egg albumin (allergens/antigen). It can be determined that, the ileum contracted thus inducing an allergic reaction, via the binding of the IgE to the FC site on the surface of the mast cells this resulted in the release of histamine from the mast cells which in turn bound onto the H1 receptors on the guinea pig ileum, and this caused the guinea pig ileum to contract (i.e. a contractile response). This response was elicited directly through an immune response by the immune system.

Additionally, because in the non-sensitised guinea pig ileum there was no response to the egg albumin, it is evident that on first exposure to egg albumin (antigen/allergen) as in this sensitised tissue, a series of events must occur in the body before the plasma cells can act by producing antigen specific IgE, also the IgE must initially be bound onto the mast cells surface receptors to stimulate a reaction, since, only previous contact with the egg albumin (antigen/allergen), would induce the synthesis of IgE and the contractile response in tissue was elicited by an immune response via the immune system. The primary response to vasodilation shows an increase in the permeability of the capillary vessels, which are located in the endothelial lining of all smooth muscle. Consequently, the permeability and vasodilation of the blood vessels results in a contractile response.

On the second exposure to egg albumin in the sensitised ileum, there was no contractile response, a reason to this was because the mast cells had already been ruptured, and through the degranulation of the mast cells, the histamine that had been released via the first exposure of the egg albumin, was bound onto the H1 receptors and the tissue would need to rest before a secondary response would be noted. However when 0.4 ml of 10-4M histamine was added a contractile response was noted. Because histamine is naturally bound onto the H1 receptors the tissue will respond. Also this histamine bound blockade was a reason why when the 0.2 ml of 10-4 M Mepyramine was added and there was no response. Mepyramine, which is an antagonist, (also acts on the H1 receptors) on the surface of the tissue therefore a response would not occur. Therefore this demonstrates that histamine produces inhibitory effects.

Adding 0.4 carbachol, which acts on the acetylcholine receptors, showed a contractile response in the guinea pig ileum. Carbacherol acts on different receptors, i.e. the neurotransmitters, which in turn elicited a contraction via the muscarinic and the choline receptors on the (guinea pig ileum) muscle cells. Therefore it seems evident that longitude and circular muscles contain and respond to excitory nerves the cells on the guinea pig ileum. It seems likely that carbachol was used to implement that the guinea pig ileum (tissue) in the experimental was still alive.

Sensitised 2 experiment, the results illustrate that neither the mepyramine, histamine nor the egg albumin showed a contraction when they were added to the tissue following the initial 0.2 and 0.4 10-4 M of histamine that was added and did show a contractile response, evidently this was because the histamine was already bound onto the H1 receptors.

When the initial 0.2 and 0.4 10-4 M of histamine was added a contractile response was noted again the response was slightly higher with the 0.4 ml of 10-4 M histamine than the 0.2 10-4 M histamine again illustrating that the higher the concentration, the larger the contraction. When the histamine is introduced to the guinea pig ileum (tissue) the natural binding elicited a contractile response via the histamine binding onto the H1 receptors on the muscle cells in the tissue. Once more when the mepyramine was added to the tissue a contraction did not occur because of the primary binding of the histamine to the H1 receptor. Thirty seconds later 0.4 ml of 10-4 M of histamine was added to the tissue and a contractile response again was not noticed.

When the tissue was exposed to egg albumin the tissue did not contract, illustrating strong evidence that once histamine was bound to the H1 receptor no other substance can bind on. Additionally it can be determined that this was an antibody mediated response directly involving the adaptive immune systems where it is highly specific on the first exposure to an antigen/egg albumin and produced memory B and T-lymphocytes consequently it made the distinction between the egg albumin evidently the immune/ inflammatory resistance was improved by a second bout of infection (second exposure to the egg albumin and it can be determined that this was an antibody mediated response directly involving the adaptive immune system.

Some of the results obtained from this experiment are similar to results that were confirmed in a study that was investigated regarding people who were sensitised to asthma phenotype. This experiment involved taking small pieces of bronchial tissue segments, which were incubated with serum (and grown in culture) from a person who, via a skin test proved positive for allergens and a person’s serum who was Non-allergic, the result was the person who was non-allergic had a lower concentration of IgE bearing cells where as the allergic person’s serum contained a high level of IgE bearing cells. Also the amount of IgE bearing cells was significantly higher in people with passively sensitised serum the mast cells revealed a 65% of IgE bearing cells The results indicated that the mast cells were the main cell type involved in IgE induced passive sensitisation.

“There are several different types of functional consequences of passive sensitization Bronchial ring segments exposed to allergic serum exhibit a contractile response to exogenous allergen, whereas segments from the same patient exposed to non-allergic serum do not. That IgE mediates this response and that the site of this reaction is mast cells located within the tissues is now well established. This evidence arises from studies in which chimeric IgE has been substituted for allergic serum (), serum has been depleted of IgE (), and anti-IgE has been substituted for allergen (). Moreover, immunohistochemical analysis of passively sensitized tissue has revealed that there is an increase in IgE-bearing cells in these tissues and that these cells are principally mast cells.”

Am. J. Respir. Crit. Care Med., Volume 161, Number 3, March 2000, S207-S210

Bibliography

Work taken from Antibodies and their uses (module) dated 6th October 2004 Professor Ellen Billet.

Hand drawn diagram taken from

http://www.emedicine.com/med/topic1101.htm#targetA

Page 119, 203-205. Rang et el, fourth edition Pharmacology

Pages111-113. Robbins et el seventh edition basic pathology

References

Current Allergy & Clinical Immunology, March 2004 Vol 17, No. 1

INTRODUCTION TO ALLERGY (title)

Dr Adrian Morris

Allergy Clinic, 112 Constantiaberg MediClinic, Cape Town

Accessed through Science direct .com and entrez pub med.

Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization. (Title)

Alessandra Mazzoni1, Howard A. Young2, Jessica H. Spitzer1, Alberto Visintin1 and David M. Segal1

Changes in afferent impulse activity of small intestine mesenteric nerves in response to antigen challenge

A. D. Nozdrachev, G. N. Akoev, L. V. Filippova, , N. O. Sherman, M. I. Lioudyno and F. N. Makarov

Laboratory of Physiology of Sensory Receptors, Pavlov Institute of Physiology, Russian Academy of Sciences, Nab. Makarova 6, St Petersburg 199034, Russia

Accepted 15 July 1999. Available online 10 November 1999.

Characterization of muscarinic receptors mediating the contraction of the urinary detrusor muscle in cynomolgus monkeys and guinea pigs.

Lai FM, Cobuzzi A, Spinelli W.

Cardiovascular/Metabolic Diseases, Wyeth-Ayerst Research, Princeton, NJ 08543-8000, USA.