Anil - Year 2 Immuno by ICSM SU

Immunology MCD Year 2 Anil Chopra

Contents Contents..........................................................................................................................1 Immunology 1 - Allergic Disease..................................................................................2 Immunology 2 – Hypersensitivity..................................................................................8 Immunology 2 – Hypersensitivity................................................................................13 Immunology 4 - Tolerance and Autoimmunity............................................................18 Immunology 5 - Immunology of Reproduction...........................................................23

Immunology 1 - Allergic Disease Anil Chopra 1. Outline the factors underlying the development of atopic/allergic diseases 2. Describe the important clinical features of asthma, hay fever, allergic eczema and anaphylaxis 3. Briefly describe the approach to investigation and management of patients with these disorders. Atopy: the immune response to a harmless antigen (allergen). Allergy: the expression of a disease caused by atopy. Common allergens include: - mites - pollen - fur - penicillin - nuts Prevalence of atopy is 50% of young adults in the UK. They do however vary in severity from mild occasional symptoms to chronic severe asthmatic attacks. The causes can be from the environmental causes: - Age - increases in children, peaks in teens, reduces in adulthood - Gender - asthma commoner in males in childhood, females in adults - Family size - commoner in small families - Infections- early life infections protect - Animals - early exposure protects - Diet - anti-oxidants, fatty acids protect …or from genetic causes: - 80% atopics have a family history. - It is shown to be polygenic – over 40 genes contribute to atopy. - IL-4 gene cluster (chromosome 5) linked to raised IgE - genes on chromosome 11q (IgE receptor) linked to atopy and asthma There are 4 types of hypersensitivity reaction:

There are a number of different types of inflammation in allergy: » Anaphylaxis & some urticarias (skin rashes) - type I hypersensitivity (IgE mediated) » Chronic urticaria & some drug allergies- type II hypersensitivity (IgG mediated) » Asthma, rhinitis (inflammation of internal areas of the nose), eczema: mixed inflammation o type I hypersensitivity (IgE mediated) o type IV hypersensitivity (chronic inflammation) Allergic Reactions take place in 2 main steps: Sensitisation: initial exposure to the allergen. This normally occurs early in life and produces a primary immune response to the antigen.

Reaction: second exposure to the antigen any time after sensitisation. This produces a secondary response induced by memory cells.

Eosinophils: make up only 2-5% of the leukocytes, are produced in the bone marrow and they reside in tissues. They have a multi-lobed nucleus and large granules of toxic proteins which they use against antigens/allergens as they are recruited in immune responses. They can also cause tissue damage. Mast Cells: mast cells are resident in tissues, and contain large granules with histamine and toxic proteins. They contain IgE on their surface which, becomes crosslinked when the come into contact with an antigen and causes the release of histamine and synthesis of some other inflammatory mediators (cytokines, prostaglandins, leukotrienes,) Dermatographic urticaria (also known as, dermatographism) is a skin disorder in which the skin becomes raised and inflamed when stroked or rubbed with a dull object due to the action of inflammation mediated by mast cells. Neutrophils: these play a role in severe and virus induced asthma. They make up 5070% of leukocytes in the blood, contain a multi-lobed nucleus and contain digestive enzymes and can reslease mediators such as cytokines, leukotrines and oxidant radicals. Asthma

Acute Inflammation » Mast cells become activated and degranulated. o Release histamine, prostaglandin, leukotrienes. o This results in the airways narrowing. Airflow limitation in patients with asthma results from one of four mechanisms related to the inflammatory process: » Airway wall oedema occurs when mediators released from inflammatory cells cause leakage of fluid from the pulmonary capillaries into the tissues surrounding the airways, resulting in mucosal thickening and swelling of the airway.

» Mucus plug formation occurs in longstanding severe asthma, and results in persistent airflow limitation due to chronic mucus secretion and the hardening of mucus plugs in the airways. » Acute bronchoconstriction occurs when smooth muscle in hyper-responsive airway walls contracts. It is a basic mechanism involved in asthma exacerbations and acute episodes of worsening symptoms and/or lung function. » Airway wall remodeling refers to structural changes in the airway that lead to irreversible airflow limitation. Chronic Inflammation Chronic inflammation of the airways results from:  Cellular infiltrate o Th2 lymphocytes, eosinophils  Smooth muscle hypertrophy  Mucus plugging  Epithelial shedding  Sub epithelial fibrosis Clinical Features of Allergy Rhinitis: the irritation and inflammation of areas around the nose. It is most prevalent in the summer (hayfever) and can also be caused by fur and house dust mites. It is characterised by runny nose (rhinorrhoea), itchy nose and eyes, nasal blockage and sneezing. Asthmatic attack: the main allergens that induce asthmatic attacks are house mites, pollen and pet fur. Asthmatics can be diagnosed by wheezing, variable peak flow and abnormal response to treatment. These can be exacerbated by viruses (cold, flu), irritants, temperature changes, emotion, exercise and certain medicines (β-blockers). Anaphylaxis: this is a severe asthmatic reaction and is uncommon. It is characterised by itchiness and swelling around mouth, pharynx, lips, wheeze, chest tightness, dyspnoea, faintness, diarrhoea & vomiting, collapse and can leave to death if severe or untreated. It is mediated by degranulation of IgE mast cells and is caused by exposure to peanuts, penicillin, NSAIDs and insect stings. Food allergies: there are a number of different allergens in food that can bring about allergic reactions. The birch/pollen allergy syndrome is characterised by sensitisation by inhalation of the birch grass/pollen which results in oral hypersensitive reactions to many foods such as fresh fruit and vegetables which only disappears on cooking. Eczema: this is characterised by a chronic itchy skin rash that is present until adult age. It can be complicated by bacterial infection.

Management of Allergies Diagnosis  Take careful history  Skin prick testing  RAST Radioallergosorbent Test (blood specific IgE): o on anti-histamines, extensive skin disease, dermatographism, baby, anaphylaxis, peanut  Total IgE  Allergen challenge  Test Lung function (asthma) Treatment  Emergency Treatment o EpiPen or Anaphylaxis kit - adrenaline, antihistamine, steroid  Allergic rhinitis o anti-histamines (sneezing, itching, rhinorrhoea) o nasal steroids (nasal blockage) o cromoglycate – a mast cell stabiliser (children, eyes)  Eczema o Emollients (moistureiser) o topical steroid cream  Venom allergies such as bee or wasp stings o Immunotherapy – more and more vaccines of the antigen are given to the patient until they are hyposensitised o single antigen o antigen used is purified  Pollen induced allergies o Immunotherapy if single allergen responsible for major symptoms o purified preparation available o SLIT – sub-lingual immunotherapy.  Asthma o Step 1. Use β2 agonist drugs as required by inhalation  salbutamol o Step 2. Inhaled steroid low - moderate dose  beclomethasone/budesonide (50-800mg per day)  fluticasone (50-400mg per day) o Step 3. Add further therapy  Add Long acting β2 agonist, leukotriene antagonist  High dose inhaled steroids - up to 2mg per day via a spacer o Step 4. Add courses of Oral Steroids  prednisolone 30mg daily for 7-14 days Prevention  Avoidance of the known allergy  Always carry a kit or EpiPen  Inform immediate family & caregivers

 Wear a MedicAlert bracelet

Immunology 2 – Hypersensitivity Anil Chopra 1. Describe the mechanisms by which IgE, antibodies, immune complexes and Tcells can cause tissue damage and inflammation (the 4 types of hypersensitivity) 2. Give examples of the clinical syndromes associated with each type of immunemediated inflammation. Inflammation Inflammation is the body’s rapid response and involves various different immune molecules including antibodies, complement, cytokines. e.t.c. as well as other immune cells moving toward areas of injury or infection. It produces  Local dilatation  Increased blood flow  Increased vascular permeability o Caused by:- C3a, C5a, Histamine, Leukotrienes, Cytokines IL-1, IL-6, IL8, IL-2, TNF, LT  Inflammatory mediators & cytokines o Cell trafficking - Chemotaxis – Chemokines o 1. Neutrophils o 2. Macrophages o 3. Lymphocytes …which results in  Heat  Pain  Redness  Swelling Tissues Injury Damage Microbe PG, LT

Mast Cells and Inflammation

Antibody

Histamine

Mast Cell

Complement activation C3b

C3a, C5a

Chemotaxis

Phagocytosis

Polymorphonuclear Leukocyte

PM N Fluid Oedema

Blood

Vascular permeability

Types of Hypersensitivity: Type I : Immediate Hypersensitivity When the antibody is primarily exposed, IgE antibodies are produced. The IgE antibodies bind to mast cells and basophils. When the patient is exposed to the antigen for the 2nd time, then a lot more IgE is produced and the antigen causes cross-bridges between the IgE molecules on the surface of mast cells. This results in degranulation and the release of histamine, tryptase and kininogenase. These cause all the immediate effects of inflammation such as:  Increased blood vessel permeability and therefore leakage.  Bronchial constriction.  Gut hypermotility. These also result in the production of new formed mediators such as:  Leukotrienes  Prostaglandins … which cause the effects of late phase inflammation. Diagnosis  History o Timing with respect to exposure o If unclear - review all exposures preceding 24 hours  Grade reaction Mild Localised angioedema & urticaria No significant impairment of breathing No features of hypotension Moderate

More widespread angioedema & urticaria Some bronchospam, Mild GI symptoms

Severe

Severe, intense bronchospasm Laryngeal oedema, severe shortness of breath, cyanosis, respiratory arrest, hypotension, cardiac arrhythmias, shock, gross GI symptoms

 Skin Prick Test  immediate wheal and flare response  Total serum IgE  Specific serum IgE – RAST  Serum/Urine tryptase  3 samples over 36 hours

Type II: Antibody-dependent Cytotoxicity  Antibodies IgG or IgM react with cell surface antigens.  This results in the activation of complement, and in turn cell lysis (death) and inflammation.  More cytotoxic cells are attracted (neutrophils, eosinophils, monocytes, NK cells) Clinical presentation depends on target tissue  Organ-specific autoimmune diseases o myasthenia gravis (Acetylcholine R Antibody) (Type 5) o glomerulonephritis (Anti-GBM Antibody) o pemphigus vulgaris (Antibody - epithelial cell cement)  Autoimmune cytopoenias (blood cell destruction) o haemolytic anaemia o thrombocytopoenia o neutropoenia  Haemolytic disease of the newborn (rhesus antibody) Diagnosis Tests for Antibody-Dependent Hypersensitivity  Test for specific autoantibodies  Organ and Non-organ specific  Use Immunofluorescence o Used to label the antibodies or antigens with a flourescent dye. Tissue slide + Serum + Fluor detector, microscope.  For identified antigens  Enzyme-Linked ImmunoSorbent Assay, or ELISA.

Type III : Immune Complex Mediated This is a very serious form of hypersensitivity formed from the formation of antigen – antibody complexes which deposit in tissue and the circulation. Complement is activated and inflammatory cells are recruited. It can lead to tissue damage and vasculitis. If the response is quick, then there is an antigen excess, if the response is very late, then there is antibody excess. In these cases, the immune complexes are small and are likely to deposit in small blood vessels.

If the response is moderate then there is more of an equal number

of antigens and antibodies. The complexes bind to the complement more efficiently and hence are cleared more efficiently.

Type IV : Delayed Cell Mediated It is called delayed cell mediated hypersensitivity because the effects take 2 – 3 days. The response is brought about by CD8+ cytotoxic T cells and CD4+ helper T cells. The CD4+ helper cells recognise and bind to the MHC class II (occasionaly I) on the surface of cells that are espressing antigen and in respones secrete:  IL(interlukin)-2: induces release of o IFN-γ o TNF-α o TNF-β (LT-lymphotoxin)  IFN-γ: o Up-regulates MHC Class II o Activates macrophages o Promotes Antibody class switching to IgG2a  TNF-α & TNF β (LT): o Activate vascular endothelium o Promote inflammation o Activate macrophages It is tested for by a contact sensitivity test.

Type V: Stimulatory Hypersensitivity This is a new proposed type of hypersensitivity and involves antibodies binding to hormone receptors which results in the gland over-secreting that hormone. E.g.  Graves Disease: Hyperthyroidism, antibody acts as TSH (thyroid stimulating hormone).

Immunology 2 – Hypersensitivity Anil Chopra 3. Describe the mechanisms by which IgE, antibodies, immune complexes and Tcells can cause tissue damage and inflammation (the 4 types of hypersensitivity) 4. Give examples of the clinical syndromes associated with each type of immunemediated inflammation. Inflammation Inflammation is the body’s rapid response and involves various different immune molecules including antibodies, complement, cytokines. e.t.c. as well as other immune cells moving toward areas of injury or infection. It produces  Local dilatation  Increased blood flow  Increased vascular permeability o Caused by:- C3a, C5a, Histamine, Leukotrienes, Cytokines IL-1, IL-6, IL8, IL-2, TNF, LT  Inflammatory mediators & cytokines o Cell trafficking - Chemotaxis – Chemokines o 1. Neutrophils o 2. Macrophages o 3. Lymphocytes …which results in  Heat  Pain  Redness  Swelling Tissues Injury Damage Microbe PG, LT

Mast Cells and Inflammation

Antibody

Histamine

Mast Cell

Complement activation C3b

C3a, C5a

Chemotaxis

Phagocytosis

Polymorphonuclear Leukocyte

PM N Fluid Oedema

Blood

Vascular permeability

More widespread angioedema & urticaria Some bronchospam, Mild GI symptoms

Severe

Severe, intense bronchospasm Laryngeal oedema, severe shortness of breath, cyanosis, respiratory arrest, hypotension, cardiac arrhythmias, shock, gross GI symptoms

 Skin Prick Test  immediate wheal and flare response  Total serum IgE  Specific serum IgE – RAST  Serum/Urine tryptase  3 samples over 36 hours

If the response is moderate then there is more of an equal number

of antigens and antibodies. The complexes bind to the complement more efficiently and hence are cleared more efficiently.

Immunology 4 - Tolerance and Autoimmunity Anil Chopra 1. To understand the concept of immunological tolerance 2. To understand the mechanisms underlying immunological tolerance 3. To understand how defects in tolerance lead to autoimmune disease There are over 70 chronic autoimmune diseases affecting 5-8% of the population (80% of which are women). The major ones are: - Rheumatoid Arthritis: 2.1 million cases 30-50,000 children, 2.1 million lost workdays - Type I diabetes: 300-500,000 cases, (123,000< 20yrs old) - Multiple Sclerosis: 250-300,000 cases (25,000 hospitalisations) - Systemic Lupus Erythematosus (SLE): 240,000 cases - Inflammatory Bowel Disease (IBD): including Crohn’s disease and ulcerative colitis)> 800,000 cases - Autoimmune thyroid disease (ATD): including Hashimoto’s and Grave’s disease: 3.5 cases/ 1000 women, 0.8 cases per 1000 men. -

They are caused by the production of antibodies and the induction of an immune response to auto-antigens: • Antibody response to cellular or matrix antigen (Type II) • Immune complex formed by antibody against soluble antigen (Type III) • T-cell mediated disease (Delayed type hypersensitivity reaction, Type IV)

Immune Response Antigens are presented to T-cells by MHC (major histocompatibility complex) antigen presenting cells.

The development of autoimmune disease can be increased by genetics. The different alleles that code for the production of HLA antigens can increase or decrease the risk of development of autoimmune diseases. Syndrome

Autoantigen

Pathology

Insulin dependent diabetes mellitus Rheumatoid arthritis

Pancreatic β-cell antigen

β-cell destruction

Unknown synovial joint antigen Joint inflammation and destruction Multiple Sclerosis Experimental Myelin Basic Protein Brain invasion by CD4+ T-cells, autoimmune encephalitis (EAE) Proteolipid protein (demyelination), weakness Myelin Oligodendrocyte glycoprotein B-cells, T-cell and NK cells all have a role in autoimmune disease.

Protective Mechanisms It has been shown that we are tolerant (unable to respond) to self antigens but this tolerance decreases with age and is specific. The tolerance is: • Acquired -involves cells of the acquired immune system and is ‘learned’. • Antigen specific • Active process in neonates the effects of which are maintained throughout life. There are a number of mechanisms in tolerance of which any can fail resulting in an autoimmune disease: Central Tolerance T-cells: Because T-cells are produce in the bone marrow but don’t mature until the get to the thymus, they can only become selective after reaching the thymus. The thymus presents the antigens on dendritic or thymic epithelial cells which then bind to immature T-cells forming a pool of immature T-cells. Only 5% of the mature T-cells in the thymus undergo positive selection and clonal expansion. These cells will be restricted from self MHC molecules and hence will be self tolerant. T- Cell repertoire before selection Negative Selection

Neglect

90%

Apoptosis Positive Selection

Apoptosis

5% Export to Periphery

Self MHC restricted, Self Tolerant, T-cell repertoire with ability to respond to pathogens

Those T-cells which have a high affinity for self antigen MHC-complexes are destroyed by apoptosis. Selection depends on the affinity of peptide antigen MHC complex: TCR interaction and mount of peptide-MHC complex.

B-cells: tolerance occurs in the bone marrow by the deletion of immature B-cells when cross-linking of the immunoglobulins on the surface of the cells.

If central tolerance fails, then a condition called APECED results: Autoimmune PolyendocrinopathyCandidiasisEctodermal Dystrophy (auto-immune polyglandular syndrome type 1) It is a rare autoimmune disease that affects all endocrine glands (thyroid, kidneys, pancreas-diabetes, gonadal failure, pernicious anaemia). It is caused by mutation of the AIRE gene which is responsible for presenting antigens to T-cells in the thymus. Most autoimmune diseases affect multi-organ systems: e.g. SLE (systemic lupus erythematosis). In SLE autoantibodies are generated against a range of broad spectrum antigens. Immune complexes activate forming deposits and causing tissue damage in a wide range of tissues.

Peripheral Tolerance Many antigens are not expressed until the immune system has matured so in order to prevent immune responses, 4 mechanisms are in place: Anergy: in order for naive T-cells to produce an immune response, they need to be co-stimulated, however most cells in the body do not have the ability to do this. B Cells’ anergy is induced by high concentrations of soluble antigen resulting in down-regulation of surface immunoglobulins. Split Tolerance: this results from the fact that in order to induce an immune response, B-cells need to be activated by T-cells. B-cells often persist in an inactive state. Ignorance: this occurs when antigenic concentration is too low in the periphery or when there is no antigen presentation (no MHC II). There are also some immunologically privileged sites where immune cells cannot normally penetrate: for example in the eye, central and peripheral nervous system and testes. In this case cells have never been tolerised against the autoantigens. - following surgery or infection sympathetic Uveitis can develop. This leads to an immune cells filtering into one eye, and hence an immure response attacking the other eye. Suppression: Autoreactive T-cells may be present but do not respond to auto-antigen because of other cell types resulting in negative signalling. Failure in peripheral tolerance can result in IPEX (Immune dysregulation, Polyendocrinopathy, Enteropathy and X-linked inheritance syndrome) - a fatal recessive disorder presenting early in childhood. It is caused by a mutation in the FOXP3 gene which encodes a transcription factor ‘scurfy’, critical for the development of regulatory T-cells. Symptoms include: • early onset insulin dependent diabetes mellitus • severe enteropathy • eczema • variable autoimmune phenomena • severe infections

Immunology 5 - Immunology of Reproduction Anil Chopra 1. Outline current theories of why the foetus is not rejected by the mother. There are a number of different hypotheses as to why implanted foetuses are not rejected by a mother. Medawar’s Hypothesis Medawar thought that it may be explained by one of (or more than one of 3 things:  Anatomical separation of the mother and the foetus  This was however proved wrong because there are 2 points of contact between the mother and the foetus.  Extravillous cytotrophoblast – this is important in early pregnancy in the process of implantation and placentation. Here, a number of different immune cells from the mother come into contact with foetal cells.  Synctiotrophoblast – the interface between the maternal blood and the synctiotrophoblast. This is important in the 2nd half of pregnancy.  Antigenic immaturity of the foetus  Neither synctiotrophoblast cells nor extravillous cytotrophoblast express MHC Class II Antigens  They do however contain some MHC class I antigens  The monomorphic class I antigens should not should not provoke an immune response by the mother.  In the decidua, there is interaction between the HLA-C, HLA-E and HLA-G antigens and NK cells (not T-cells). This results in the facilitation of invasion of the trophoblast.  The decidual NK cells produce a number of factors which facilitate trophoblast invasion including cytokines (IFNγ, IL-10, TGF-β1, TIMP-1) chemokines (IL-8, IP-10) and angiogenic factors (VEGF, PLGF).  It has been shown the level of HLA-G secreted by the trophoblast cells correlates with implantation success.  Abnormal expression of HLA-G antigens can lead to complications in pregnancy – pre-eclampsia (hypertension in pregnancy due to increased proteins in the blood).  There are 2 types of HLA-C antigen that are expressed by the trophoblast cells which bind to the KIR (Killer Ig-like Receptor) on the NK cells. It they express HLA-C1 then the NK cells are activated (esp. if the NK cell express KIR-B) and if they express HLA-C2 then NK cells are not activated (esp. if the NK cells express KIR-A).

 Maternal immunological inertness  Foetal leukocytes travel into the maternal blood.  Whilst the mother can make antibodies to the foetal leukocytes and an immune response occurs, it appears to have no effect on the baby.  Maternal IgG antibodies are transported across the placenta into foetal blood however the antibodies that were produced against the foetal leukocytes cannot get into the foetal blood because of complement regulatory proteins on trophoblast cells e.g DAF CD46.  Rhesus D antibodies can cross the placenta!  It has been shown that antibody mediated response to foetal HLA is normal but cell mediated responses are depressed.

Evidence for this includes:  Temporary remission of Rheumatoid Arthritis (Th1 mediated) during pregnancy  Diseases caused by intracellular pathogens (e.g. Herpes and Malaria) are exacerbated by pregnancy  Systemic Lupus Erythematosis (SLE) (Th2 mediated) gets worse during pregnancy  In abnormal pregnancy, Th1 mediated immunity is not suppressed.

• Anatomical separation of the mother and the fetus No • Antigenic immaturity of the fetus HLA-G/HLA-E/HLA-C • Maternal immunological inertness (Th1/Th2 shift – decreased cell mediated immunity but normal antibody production