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Toxic Responses of the Immune System

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Hayley Beaton

on 15 April 2011

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Transcript of Toxic Responses of the Immune System

Toxic Responses of the Immune System Overview Immunosuppression Immunostimulation Assays Many therapeutic agents can have major immunomodulatory effects specifically in relation to immunosuppression. When the immune system is suppressed, the most severe consequences will be an increase in the incidence and severity of infections and/or an increase in the incidence and progression of malignancies or cancer. A variety of these therapeutic agents will be discussed further with regards to their ability to cause a deficiency within the immune system.

Organophosphates include malathion, parathion, methyl parathion
Malathion can suppress humoral immunity. In vitro exposure of human cells to malathion can cause decreased lymphoproliferative responses, suppressed CTL generation and may also cause apoptosis in thymocytes.
Parathion is more acutely toxic than malathion. This pesticide suppresses both humoral and CMI. Following exposure thymic atrophy and lowered DTH responses have been documented. Suppression of lymphoproliferative responses and an increased susceptibility to pathogenic infections has also been reported. In vitro experiments have shown that parathion reduces CMI, IL-2 production and proliferative responses in human lymphocytes.
Polyaromatic Hydrocarbons

Polyaromatic Hydrocarbons (PAH’s) are a widespread group of organic pollutant and are recognised to be a group of potent immunosuppressant. Effects on immune system development, humoral immunity and on host responses have been documented after exposure to PAH’s.
The most common immunotoxic member is DMBA (7,12dimeythlbenz(a)anthracene). Its immunotoxicity is mediated through two mechanisms. The first mechanism is the activation of the Aryl Hydrocarbon Receptor which is a specific cytosolic receptor, The second is by their ability to generate a reactive intermediate via cytochrome P450 enzymes. Certain types of immunocompetent cells, most notably the macrophage, have enough metabolic capacity to activate PAH’s.

DMBA significantly suppresses not only PFC (plaque-forming cells) but also NK cell activity, Cytotoxic T-Lymphocyte responses, Delayed-type hypersensitivity responses and lymphoproliferative effects. DMBA exposure results in long-lasting suppression of humoral and cell mediated immunity and also tumour resistance mechanisms in mice.
They also exert their immunotoxic effects by inducing apoptosis of pre/pro B-cells which are the precursors of mature B-cells. Direct cell-to-cell contact with bone marrow stromal cells is required to deliver the apoptotic signal. DMBA (7,12-dimethylbenzanthracene) a protein that is derived from the bone marrow in order to elicit its immunotoxic effects.

Arsenic is a primary immunosuppressant that targets both macrophages and T-cells. Antigen processing and presentation is the specific target of arsenic in macrophages. A decrease in the expression of T-cell surface molecules leads to antiproliferative effects on T-cells and thus immunosuppression. This antiproliferative effect of arsenic on T-cells can be reversed by the exogenous addition of IL-2, IL-5 and IL-6.

Lead has shown to cause a decrease in resistance to the bacterial pathogens S. Typhimurium and Escherichia Coli. Lead exerts its immunomodulatory effects by lowering the amount of antibodies and also suppresses humoral immunity by decreasing IgA levels within the immune system. It also has been consistently shown to suppress IgM levels which is as a result of an affecting on macrophage function. It also alters immune recognition through an alteration in the ability of macrophages to process and present antigen-antigen primed T-cells. Drugs of abuse

Cannabinoids are a family of more than 60 structurally related molecules, which constitute the active ingredients of marijuana and exert their immunotoxicity by selectively suppressing T cell and macrophage function. The mechanism of action is still unclear but it seems to be partly mediated by specific cannabinoid receptors, termed CB1 and CB2. Both CB1 and CB2 belong to the G-protein coupled receptor superfamily.

The binding of cannabinoid ligands to cannabinoid receptors results in two independent events. The first is the inhibition of adenylate cyclase, the enzyme that converts ATP to cAMP. The downstream consequence is an inhibition of the cAMP signaling cascade as evidenced by decreased protein kinase A activity, reduced DNA binding by cAMP response element-binding proteins, and reduced transcription of cAMP responsive genes. The second consequence is a rapid induction of intracellular calcium, through the opening of calcium channels. Elevation of intracellular calcium prior to antigenic stimulation renders T cells unresponsive to antigenic stimulation. Drugs of abuse

Opiates, such as morphine, heroin & fentanyl, can also cause transient immune system depression though the exact mechanism of action remains unclear. Morphine is capable of affecting both the innate and adaptive immune response. It is thought that these opiates indirectly elevate adrenal corticosteroid levels by stimulating the hypothalamic-pituitary-adrenal neuraxis. In vitro experiments have been undertaken to understand the effects of morphine on immune system depression and the results show that high doses of morphine transiently suppress NK cells function within the immune system.

Cocaine can trigger changes in many neuroendocrine factors which can suppress immune function. Cocaine stimulates the hypothalamic-pituitary-adrenal neuraxis which causes an elevation in plasma corticosterone which in turn leads to suppression of immune function.

Ethanol is another example of a drug which may cause immune suppression. Most of the evidence for the cause of immune suppression points to elevated levels of serum corticosterone at doses which are capable of causing immunosuppression.

A glucocorticoid antagonist e.g. RU-486 is capable of reversing this suppression of immune function.
Natural Hormone

Glucocorticoids are hormones whose immunosuppressive properties have been known for many years. Their mechanism of action is thought to involve the binding of glucocorticoids to a cytosolic glucocorticoid receptor which induces the receptor to function as a ligand-activated transcription factor that undergoes homodimerisation and DNA binding to glucocoricoid response elements (GRE) in the regulatory regions of glucocorticoid responsive genes. Depending on the gene GRE can either positively or negatively regulate transcription.
Following binding to the cytosolic receptor these steroid hormones produce lymphoid cell depletion and lymphopenia associated with a decrease in monocytes and eosinophils. Corticosteroids also induce apoptosis to which T-cells are particularly sensitive targets, they cause inhibition of humoral mediated immunity through their affects on T-cells. Their affect on B-cells still remains unclear. They also function to suppress macrophage accessory cell function, the production of IL-1 from macrophages and the subsequent generation of IL-2 by T-cells. These chemicals also induce transcription of the endogenous inhibitor of the NF-αβ which results in the suppression of inflammatory cytokine production. In general, glucocorticoids cause immunodeficiency by suppressing CTL responses, NK cell activity and lymphoproliferation. Synthetic Hormones

Estrogens e.g, diethylstilbestrol is a synthetic non-steroidal compound containing estrogenic activity. Exposure to diethylstilbestrol results in changes in both cell mediated immunity and macrophage function. The thymus is particularly targeted by this compound and leads to thymic depletion and alterations in the T-cell maturation process. At high concentration this synthetic hormone decreases both T- and B-cell populations leading to a deficiency within the immune system. Immunostimulation:
In toxicology, immune stimulation can be defined as the unwanted “switching on” of immune system components in response to toxins. This occurs when the immune system recognises toxins as non-self and an immune response is triggered. There are a huge range of toxins varying in nature that can stimulate the immune response and cause hypersensitivity reactions and autoimmunity. These include a number of drugs, foods such as nuts and shellfish, plant allergens (for example urushiol- chemical in both poison ivy and poison oak), industrial chemicals and other chemicals such as apitoxin, the main component of bee sting. Mechanism:
The way in which toxins interact with the immune system and induce a stimulatory response is quite complex. For the immune system to launch an attack on a substance it must be recognised as non-self. Most toxic chemicals are too small to be recognised by the immune system. Therefore if they are to trigger an immune response they must act as haptens. Haptens have the ability to interact with and bind to proteins within the body thus forming a hapten-carrier complex. This interaction changes the protein so that it is no longer recognised as self and thus the immune response begins. The hapten-carrier complex is the earliest theory put forward explaining how chemicals can cause an immune response. Recent Research:
In recent years further research has been put into determining whether there are other ways aside from the hapten-carrier complex theory that drugs can interact with the immune system to trigger a response. In 2002, a Swiss scientist called Pichler believed that some drugs can reversibly bind to receptors on T-Cells (TCRs) and MHC receptors on other cells. This interaction may occur due to pharmacological similarities between these receptors and the receptors certain drugs are developed to target, hence why this interaction has been given the name “the p-i concept” (pharmacological interaction with immune receptors). Drugs that have been proven to have this effect and thus cause hypersensitivity reactions include carbamazapine (anticonvulsant), lidocaine (anaesthetic) and sulfamethoxazole (antibiotic). Autoimmunity and Hypersensitivity:
Immune stimulation can cause autoimmunity and hypersensitivity reactions. Autoimmunity can be defined as the loss of the ability of the immune system to distinguish between self and non self. The result is unwanted damage to healthy tissues and organs and thus the development of autoimmune diseases such as Multiple Sclerosis and Rheumatoid Arthritis. Although it is known that immune stimulation can cause autoimmunity much more research is needed in this field.
Hypersensitivity is the primary example of toxin-induced immune stimulation. Hypersensitivity can be defined as an exaggerated immune response to a foreign antigen that may cause damage to tissues and organs. Hypersensitivity can be subdivided into 4 main categories based on the mechanism of immune response. Types I-III are caused by humoral immunity responses and type IV is caused by cell-mediated immunity responses. Type I Hypersensitivity:
Type I hypersensitivity reactions result from a humoral immunity response and is immediate (seconds/minutes). This type of hypersensitivity occurs when substances such as chemicals or drugs act as haptens thus forming a hapten-carrier complex that then binds to specific target cells. Once the hapten-carrier complex interacts with the cell it is no longer recognised as self and thus B-cells are stimulated to proliferate and produce antibodies. These B-cells then release the antibody IgE which binds to and interacts with other immune cells such as mast cells and basophils. These cells are now said to be sensitized to the specific hapten-carrier complex. When these cells come in contact with this specific complex again, they will immediately recognise and launch an immune response. They do so by degranulating, thus releasing histamine and other substances, causing an inflammatory response and possible tissue damage. The organs that are mainly effected during a type I reaction are the gastrointestinal tract, the skin, the lungs and blood vessels. Type I continued:
Food allergies are examples of type I reactions. In most cases this causes nausea, vomiting, diarrhoea, itchiness of the skin and rash formation. However in more severe cases it may result in anaphylactic shock. This allergic response can be fatal in some cases as the airways tighten due to smooth muscle spasms making it difficult to breath. Blood pressure also falls dramatically. Adrenaline is the antidote for this type of allergic reaction. An important point to note is that there can be a genetic component to some food allergies. Other examples of type I reactions include hayfever and asthma.
Other substances that cause type I reactions are penicillin, cephalosporins, sulphonamides, NSAIDs such as ibuprofen and aspirin, morphine and chemical in the stings of insects such as apitoxin in bee sting. Type II Hypersensitivity:
This type of hypersensitivity occurs due to a humoral immunity response and is nonimmediate. The mechanism is the same as type I however the main antibodies involved in this type of response are IgM and IgG. In this case the “target organs” are the cells of the blood. This type of response causes cytotoxicity of the cells affected. It can be cause by penicillin coating red blood cells or sulphonamides binding to white blood cells. An example of a disease caused by chemicals that induced type II hypersensitivity is haemolytic anaemia. Diagram shows a patient with jaundice, a symptom of haemolytic anaemia. Type III:
Type III hypersensitivity is also due to a humoral immunity response caused by chemicals interacting with the immune system. It is non-immediate so its affects aren’t seen until hours/days after exposure. The mechanism for type III is the same as type I and type II however the antibody produced against a particular hapten-carrier complex is IgG. These antibodies are soluble and are easily deposited in small capillary beds for example in the skin and in the glomerular regions of the kidneys. This type of hypersensitivity results in the development of certain disease states such as serum sickness and immune complex glomerulonephitis. Type IV:
Type IV hypersensitivity is much different than the previous 3 types. Type IV is caused by cell mediated immunity, not humoral immunity. It takes between 24 and 48 hours before the first signs of a type IV hypersensitivity reaction are noted. Hence why this type of reaction is also known as delayed hypersensitivity. T-cells are activated either directly (p-i concept) through TCRs or indirectly by hapten-carrier complexes. T-cell activation can cause the stimulation of a number of other immune cells such as monocytes, eosinophils and neutrophils. These all cause an inflammatory response by releasing meditators which causes urticaria (raised itchy patches) and angioedema (fluid build up and swelling). Cytotoxic T-cells (CD8+) can cause an inflammatory response also by releasing cytokines such as IL-5 and chemokines such as IL-8.
Common type IV reactions are contact dermatitis and eczema. In contact dermatitis, an inflammatory response is triggered when the skin comes in direct contact with substances such as urushiol (the chemical in poison ivy), cosmetics, solvents, nickel-containing jewellery and topical medications such as betamethasone and iodine. Penicillin Reaction Penicillin Reaction Poison Oak Allergic Reaction Structure of Estrogen The preliminary screen of possible immunotoxicity should be included in standard toxicity studies. There are several observations that should be noted during these tests:
• Changes in haematology, for example lymphocytopenia or lymphocytosis; leukocytopenia or leukocytosis; granulocytopenia or granulocytosis.
• Differences in histology and/or weights of organs that are part of the immune system (bone marrow, thymus, lymph nodes, etc).
• Alterations in serum globulins that take place without other causes, for instance effects on the kidneys or liver can be a sign of change.
• Higher frequency of infections.
• Augmented incidence of tumours – can be taken as an indication of immunosuppression if other causes (hormonal effects, genotoxicity, liver enzyme induction) are not present.
Like other standard toxicity studies, the doses, severity, significance, duration, reversibility, and mechanism of action of the drug should be taken into account. Unusual findings from these studies would prompt additional immunotoxicity studies. Host Resistance Studies

Host resistance studies consist of challenging sets of rats or mice that have been treated with various doses of the drug to assorted concentrations of a pathogen (viral, fungal, bacterial, parasitic) or tumour cells. Infection of the pathogen or tumour burden detected in control animals versus animals that have been treated with the drug is used to establish whether the drug alters host resistance. A wide range of pathogen models, as well as tumour models, have been used in these studies. These assays can also offer knowledge on the susceptibility of the host to certain types of tumour cells or infectious agents, which can affect the risk management plan. Host resistance assays also play a key role in classifying which cell types are affected by the drug. In addition to this, host resistance assays are associated with the innate immune response system for which adaptive immune response assays have not yet been created. Macrophage/Neutrophil Function

Macrophage/neutrophil function assays can be conducted in vitro or ex vivo. These tests evaluate the function of macrophage or neutrophil cells that have been exposed to the drug in question. In vivo assays can also be utilised to measure the effects of the drug on the reticuloendothelial cells to phagocytise through fluorescently or radioactively labelled targets. Introduction

Side effects of drugs on the immune system must be assessed as part of standard drug development protocols. Immunotoxicity includes a number of adverse effects, such as immunosuppression or enhancement. Immunosuppression can give rise to decrease host resistance to tumour cells or infectious agents. Enhancement of the immune system can amplify hypersensitivity or autoimmune disorders. Sometimes, drugs or drug-protein complexes can be identified as foreign and trigger anti-drug responses. Therefore, most immunotoxicity studies are focused on the evaluation of a drug’s potential of stimulating either contact sensitisation or immunosuppression. There are two groups of drugs that are associated with immunosuppression or enhancement. The first group of drugs includes those that are meant to mediate immune activity for therapeutic purposes, such as those to counter rejection of organ transplantations, but cause unintended immunosuppression due to exaggerated pharmacodynamics. The second group of drugs are those that are not meant to affect immune activity but result in immunotoxicity, for example those that cause apoptosis or necrosis of immune cells or those that interact with receptors that are in common with both the target cells and the non-target immune cells. T-cell dependent antibody response (TDAR)

TDAR is used to determine potential immunotoxicity, particularly immunosuppression, caused by drugs. It evaluates the immune response (antibody production) to a proposed antigen (keyhole limpet hemocyanin (KLH)) in mice or rats that have been administered with a chemical (new drug). It is necessary for numerous effector cells, such as T-cells, B-cells, antigen-presenting cells, to participate in TDAR to generate an antigen-specific antibody response. Therefore, changes in antibody production levels to the particular antigen may indicate effects on one or all of the cells included in TDAR. ELISA can be used to measure antibody response. Immunophenotyping

Immunophenotyping is the classification and/or specification of leukocyte subsets by employing the use of antibodies. It is usually carried out by immunohistochemistry (IHC) or by flow cytometric analysis. Flow cytometry can be used to determine antigen-specific immune reactions of lymphocytes. However, an advantage of IHC is that tissues can be examined retrospective of standard toxicity studies if indications of immunotoxicity ha
ve been detected. Furthermore, alterations in cell types within a particular part of the lymphoid tissue can be examined. On the other hand, the magnitude of staining and quantification of leukocytes is a lot harder with IHC than with flow cytometry. Immunophenotyping can be readily incorporated into standard repeat dose toxicity studies. Natural Killer Cell Activity Assays

NK cell activity assays can be carried out if standard toxicity studies show augmented rates of viral infections, or if a change in number of cells has been detected in immunophenotyping. These assays are usually conducted ex vivo, where the tissue or blood acquired from animals has been administered with the drug. NK cell preparations and target cells that have been labelled with radioactive chromium (51Cr) are co-incubated, and the amount of killing of target cells is measured using a flow cytometer.
Assays to Measure Cell-mediated Immunity

Assays to measure cell-mediated immunity are not as established as assays that are used to measure antibody response. Assays to measure cell-mediated immunity are in vivo assays where antigens are administered to trigger sensitisation. The endpoint is the capacity of the drug to mediate the response to challenged. Delayed-type hypersensitivity (DTH) is commonly observed in these assays. However, care must be taken so that DTH is not mistaken for Arthus reaction (complement and antibody mediated reaction).
Joyce Lam Hayley Beaton Anne Maguire Ciaran Dunne References Image Sources

•Background Cell Image: topnews.net.nz

•Main Body: http://www.cancer.gov/PublishedContent/Images/images/documents/02853881-1148-4541-b2b6-e8ecd3e50c6f/cancer21.jpg

•T-cell: http://images.the-scientist.com/content/figures/0890-3670-050718-15-1-2.jpg

•T-cell 2: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/ClassIpath.gif

•T-cell 3: http://www.karger.com/gazette/65/weyand/images/weyand_3_p.jpg *

•Spleen: http://www.acm.uiuc.edu/sigbio/project/updated-lymphatic/spleen_cs.jpg

•Bone Marrow: http://en.wikipedia.org/wiki/File:Gray72-en.svg *

•Immune Cell Summary: http://lpi.oregonstate.edu/sp-su98/images/immune1.gif

•Alcohol: irishpirates.org

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•Assay Flow Chart:http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002851.pdf References

•The Encyclopedia of Toxicology, 2nd Edition, Wexler, Peyster, Academic Press 2005.

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Immunotoxicity Studies for Human Pharmaceuticals

•http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60761-401-2_11 Immunotoxicity Testing : Methods and Protocols.
The T-Dependent Antibody Response to Keyhole Limpet Hemocyanin in Rodents
By: Lisa M. Plitnick1, Danuta J. Herzyk












•www.medicinenet.com •The immune system consists of the innate immunity and the adaptive immunity. The innate immune system consists of the physical barriers; the biggest being the skin (and the secreted sebum), others being the mucosa in the bronchus, the stomach and GIT, (the acidity and natural flora). The tears act as a physical-chemical barrier also, as the tears have salinity. The innate immunity also consists of mast cells, phagocytes (macrophages, neutrophils, dendritic cells) basophils & eosinophils, natural killer cells and γδ T cells.In our group we are focusing on the cells of the immune system and how they are hyper-/hypo- sensitised. B-Cells are the main cells of the humoral immune system. They originate in the bone marrow (hence: B-Cells). Their functions are to produce antibodies and antigens to previously encountered pathogens; B-Cells are also the presenting cells which then develop later on into memory B-Cells – essential for the adaptive immune system. Other cells include Plasma B-Cells, B-1 cells (found in the peritoneal and pleural cavities), B-2 cells, Marginal-zone B-Cells and Follicular B Cells. B Cells do not require the antigen to be bound to any other protein; just the antigen itself suffices T-Cells are the main cells of the cell-mediated immune system (Consisting of Helper T-Cells, Cytotoxic cells, Memory T-Cells, Regulatory T-Cells, NK cells and γδ T-Cells). They originate in the thymus (hence: T-Cells). Their functions are to bind to the antigens; which are presented by the B-Cells. T-Cells become activated when another antigen is encountered – i.e. it double checks if the antigen was present. All T-Cells have CD3 receptor complexes; CD8 antigens are present on Cytotoxic Cells and TS-Cells. CD8 respond to antigens on MHC (class 1) proteins. CD4 antigens are found on T-Helper cells and respond to antigens on MHC (class2) proteins. The spleen contains ‘White pulp’ and ‘Red pulp’. ‘White pulp’ acts as storage/reserve for the humoral and cell mediated immune response cells; storing B-Cells and T-Cells. ‘Red pulp’ acts as a storage for monocytes. The bone marrow is the source of B-Cells. B-cells are derived from the ‘yellow’ bone marrow (the ‘red’ bone marrow produces RBC). Once B-Cells are mature, they can cross the bone-marrow barrier; permissible by the presence of membrane proteins. To summarise the cells, and their mechanisms pictorially:
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