IgE receptors

Question 1

1. why is it important to understand the mechanism of hypersensitivity?
2. which 2 cells mediate the allergic response?
3. where do these cells reside?
4. what type of receptors do these cells express?

Answer 1

1. to therapeutically prevent chronic disease associated with hypersensitivity
2. mast cells and basophils
3. mast cells reside in tissues; basophils circulate in the blood stream
4. IgE

Question 2

Systemic Anaphylaxis

1. what type of cell mounts systemic anaphylaxis?
2. what is the route of entry of allergens?
3. name 5 responses
4. why is it life threatening?

Answer 2

1. basophils
2. Intravenous, or rapid absorption from the gut
3. oedema, increased vascular permeability, tracheal occlusion, circulatory collapse and death
4. dramatic decrease in BP

Question 3

Asthma

1. how does asthma differ from hayfever?
2. name 3 asthmatic responses to an allergen
3. what occurs during the early phase?
4. what is the severity of the early phase dependent upon?
5. what occurs during the late phase?
6. name 2 things that can cause chronic asthma.

Answer 3

1. both upper and lower respiratory tracts are affected
2. bronchial construction, increased mucous production and airway inflammation
3. airway becomes obstructed and inflamed due to the release of histamine and prostaglandins.
4. the number of mast cells in the bronchial smooth muscle
5. cytokines are released, which mediates the infiltration of leukocytes. The infiltration of eosinophils is particularly damaging because they release tissue damaging factors.
6. repeated exposure to allergen, and certain infections can exacerbate symptoms

Question 4

1. which cells take up and process the allergen?
2. where do these cells migrate to?
3. how do these cells present the allergen to T cells?
4. which IL is needed for the T cell to be converted into a TH2 cell?
5. how does the TH2 cell activate the B cell?
6. what ILs are needed for the B cell undergo a class switch recombination reaction? What does this mean?

Answer 4

1. dendritic cells
2. to regional lymph gland or act locally
3. via MHC class II receptors. Also mediated by jagged on dendritic cell binding to notch on T cell
4. IL-4
5. CD40 and CD86 on B cell must be activated simultaneously
6. IL-4 and IL-13

Question 5

1. what does the ease of allergen binding to IgE depend upon?
2. what do active mast cells do? (2)
3. what do prostaglandins and histamines mediate? (5)
4. why is the late phase delayed?
5. what do basophils do?
6. what do eosinophils do? (2)
7. what do neutrphils do?

Answer 5

1. the integrity of the epithelial barrier
2. degranulate - vesicles fuse with the PM to release histamine and proteases, and produce and release lipid mediators
3. bronchial constriction, goblet cell activation, increased vascular permeability, T cell activation and recruitment and act on local sensory neurons to produce hyperalgesia
4. because cytokines and chemokines, that promote the infiltration of other immune system cells, must be synthesised de novo
5. produce IL-13, which further activates goblet cells
6. secrete MMP (peptidase) which breaks down collagen, leading to scar formation which further compromises the epithelial barrier; secrete eosinophil basic proteins which are toxic to epithelial cells
7. secrete damaging proteins such as elastase (compromises intgrity of connective tissue)

Question 6

Describe the airway remodelling that occurs in asthmatics. (5)

* what does IL-5 mediate?

Answer 6

1. cytokines induce muscle hypertrophy and hyperplasia
2. airway is obstructed by mucous
3. epithelial barrier integrity is compromised
4. T cells and B cells become resident and proliferates - positive feedback (more IgE produced)
5. increased no of mast cells

* infiltration of eosinophils so they become resident in the lungs

Question 7

describe 6 treatments of asthma

Answer 7

1. anti leukotrines
2. corticosteroids
3. β2AR agonists
4. antihistamines
5. avoidance of allergen
6. monoclonal antibodies

Question 8

1. describe the structure of the FcεR1 receptor
2. describe the structure of the IgE antibody
3. why is the interraction between IgE and receptor considered to be Irreversible?
4. what is the synthesis of the receptor mediated by?
5. give an example of a protein that is upreglulaed by mast cell activation, and what this protein does.

Answer 8

1. tetramer consisting of α, β and 2γ subunits. α subunit contains the IgE binding site and a TM domain. β and γ subunits contain ITAM motifs
2. it contains an extra domain on the heavy chain making them bent.
3. the bent IgE structure and asymmetrical binding to the receptor means that the IgE can switch between the Ig domains. As it dissociates from one IgE domain, it binds to the other domain.
4. IL-4 promotes the synthesis of the receptor.
5. P-selectin, it is an adhesion molecule which is expressed on WBCs to facilliate their infiltration.

Question 9

1. what is required for downstream signalling events? What does this mean for the antigen?
2. what protein is activated first?
3. describe the process of the activation of further adaptor proteins
4. What does PLCγ mediate?
5. what fo GRB2 and GADS mediate?
6. name a Ca mediated transcription factor. Name 2 things that they induce.
7. how does Phospholipase AZ mediate the production of lipid mediators.
8. Which proteins are involved in the complimentary amplification cascade?

Answer 9

1. receptor cross linking. The antigen must be polyvalent (ie. must have 2 recognition domains to bind to 2 IgE simulataneously)
2. LYN Kinase
3. Lyn phosphorylates ITAM motifs of the receptor creating docking sites for Lyn and SYK.SYK autophosphorylates itselfSYK and Lyn phosphorylate LATLAT co-ordinates activation of further signalling molecules
4. produces IP3 and DAG from PIP2. Both promote Ca²⁺ release leading to degranulation
5. MAPK activation
6. NFAT. induce the expression of pro-inflammatory cytokines and activate phospholipase AZ
7. breaks down PM phospholipids to arachidonate. COX enzymes break down arachidonate into inflammatory mediators
8. FYN phosphorylates NTAL

Question 10

1. how have haemopoetic stem cells been used to discover proteins involved in signalling pathways
2. What factor is vital for mast cell survival
3. how can organisms lacking this factor be used to discover signalling pathways

Answer 10

1. taking them from organisms that are deficient in a specific prorein and subjecting them to specific conditions so they differentiate into mast cells
2. Stem Cell Factor
3. injecting bone marrow derived mast cells that lack a particular protein into SCF deficient animals.

Question 11

1. where are IP₃ gated channels located?
2. what happens upon ER store depletion.
3. describe the 2 calcium signals upon mast cell activation
4. what does the removal of EC calcium show about the first signal.

Answer 11

1. ER membrane
2. activation of PM CRAC channels to amplify and prolong the calcium signal
3. first is small and transient, caused by the release from ER storessecond is large and sustained, caused by the influx from the EC space
4. that it is insufficient to cause degranulation and gene transcription.

Question 12

1. What is SCID?
2. how did they observe the effects of SCID on calcium signalling?
3. Under what conditions were the above performed?
4. what were the results
5. what was used to determined the level of the SCID defect?
6. what were the results of the above, and what does this imply?

Answer 12

1. Severe Combined Immunodeficiency (no functioning immune system)
2. isolated T cells from SCID patients.
3. in the absence and presence of EC calcium
4. when agonist applied in the absence of EC calcium, both WT and SCID cells has a small transient Ca signal.

When agonist applied in the presence of EC calcium, WT cells produced a second, larger Ca signal that was absent on SCID T cells

5. thapsigargin. It is a Ca-ATPase inhibitor that causes agonist independent store depletion
6. normal patients had a Tg induced second signal. This was abolished in SCID patients. This implies that the SCID defect lies at the level of ER activation of CRAC.

Question 13

1. Under what conditions was an RNAi screen performed?
2. what type of flies did they look for?
3. What protein was found to be critical for the second calcium signal
4. what did structural analysis of this protein reveal?

Answer 13

1. Thapsigargin store depletion and add back of EC calcium
2. flies that had the same phenotype as the SCID T cells
3. STIM
4. posesses 2 (calcium sensing) EF hands, a TM domain and coiled coils. This is not a channel forming structure.

Question 14

1. what technique was used to show the localisation if STIM under resting and Ca depletion conditions
2. What were the proteins tagged with?
3. where was STIM found at rest?
4. where was STIM found upon store depletion?

Answer 14

1. live cell fluorescence imaging
2. ER marker tagged with RFP; STIM tagged with GFP
3. rougly associated with ER
4. associated with PM

Question 15

1. what did genetic analysis of SCID patients reveal?
2. in the study of this gene, what was used as a indicator of calcium signalling?
3. what happened in control cells upon thapsigargin treatment
4. what happened in cells expressing the mutant gene upon thapsigargin treatment
5. what did structural analysis of this newly identified protein reveal?

Answer 15

1. a single mutation in the Orai1 gene
2. translocation of NF-kB-GFP to the nucleus
3. NF-kB translocated to the nucleus
4. NF-kB remained in the cytosol
5. it has 4 potential TM domains, which is expected of a channel forming protein.

Question 16

1. describe the model of CRAC activation by STIM
2. what is the phenotype of orai KO mice?

Answer 16

• EF hands of STIM are exposed to the lumenal side of the ER. They bind to Ca in the store
• upon store depletion, Ca no longer binds to STIM, therefore its structure changes
• the conformational change causes it to cluster and make contact with Orai1
• clustering of STIM mediates the formation of the Orai1 tetramer to prodcue a functional channel.
  2. severe immunodeficiency. produce a normal amount of mast cells, but signalling is defective. no histamine is produced.

Question 17

name 4 potential novel therapies for hypersensitivity reactions, and any problems associated with them.

Answer 17

1. Syk kinase inhibitors - likely to cause widespread immunodeficiency as they are found and mediate signalling in other immune system cells
2. SCF receptor antagonists/SCF blocking antibodies - shouldnt be given to children as SCF is required during development
3. anti-IgE antibodies - expensive
4. Orai inhbitors - immunodeficiency; use dependent inhibitors?

Question 18

1. How does IgE positively regulate the surface expression of FcεR1
2. give 2 piece of evidence that shows that highly cytokinergic IgE clones can lead to antigen independent receptor activation.
3. why is the finding that each of the receptor sites have an intrinsic affinity much lower than the receptors overall affinity important?

Answer 18

1. stabilises IgE at the surface by preventing degradation whilst maintaining basal levels, leading to the progressive accumulation of receptors at the surface
2. Incubation of receptors with these clones caused receptor aggregation without antigen crosslinking

incubation with these clones also produced antigen independent histamine synthesis

3. important for the development of therapeutic strategies to interfere with the receptor-IgE interraction

Question 19

1. In studies into the regulation of CRAC channels by STIM, what were CRAC inhibitors used for?
2. What were the results of using the CRAC inhibitors
3. what type of STIM mutants were expressed in cells?
4. what was used to test for apoptosis?
5. what were the effects of expressing the mutant STIM?
6. where was the mutant STIM localised prior thapsigargin treatment?

Answer 19

1. to see if Tg independent influx was mediated by CRAC
2. inhibited the Tg independent influx - this influx is mediated by CRAC
3. STIM constructs containing mutations in the EF hands
4. annexin V
5. increased Tg independent ca influx and increased apoptosis
6. PM