Chemokines in the Lung Flashcards
Recall the differences in the histopathology of asthma and COPD
The differences in lung inflammatory cells seen in pathology is due to the different chemoattractants present. Define a chemoattractant.
Any substance that can cause a cell to move.
Describe three types of chemoattractant and give examples.
- Chemokine: small proteins that causes cells to move (CXCL8)
- Lipids: Leukotriene B4 - potent neutrophil chemoattractant
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Peptides:
- formyl-met-leu-phe (fMLP) bacterial cell wall peptide - neutrophil chemoattractant
- N-Ac-pro-gly-pro (PGP) matrix breakdown peptide - neutrophils to clear up debri
Explain the signalling mechanism of chemokine receptors
Chemokines act on G-protein coupled receptors.
Ligand binds, GTP activates the a-subunit.
a-subunit activates PLC by hydrolysis of the GTP.
PLC breaksdown PIP2 into IP3 and DAG
DAG activates PKC
IP3 acts on receptor of ER releasing Ca2+ into cytoplasm.
Ca2+ leads to actin rearrangement and movement of cell.
B/g subunit activates PI3K
PI3K upregaultes adhesion molecules on cell surface
PI3K can also cause actin rearrangement.
Explain the concept of chemokine receptor redundancy
Understanding of chemokines where:
- More than one chemokine can bind to a single receptor (CXC9, 10, 11 -> CXCR3)
- A single chemokine can bind to many receptors (CCL5 -> CCR1, 3, 5)
- Each cell has more than one type of receptor (Neutrophils have CXCR1 and CXCR2)
Neutrophils: CXCR1 thought to be involved in chemotaxis and CXCR2 in degranulation
Describe three assumptions made on redundancy
Redundancy assumes:
- Different chemokines are equipotent at a single receptor (CXCL11 >> CXCL9/10 at CXCR3)
- Different chemokines have the same function at a single receptor
- Different receptors on a single cell have the same function (neutrophil CXCR1 and CXCR2; chemotaxis and degranulation respectively)
Describe and explain the use of chemotaxis assays (2) to measure migration of cells.
- Boyden chamber: A filter paper with fixed holes is put in between two chambers. The hole size will vary depending on what cell is observed (PBMC/monocytes need 8um whilst neutrophils can go through smaller holes as they can change shape more easily). Top chamber will contain the cells whilst the bottom contains the chemoattractant. The filter is fixed and stained and cells are counted on microscope.
2. Transwell Systems: Wells are used to grow endothelial cells on one side and epithelial cells on the other. Cell movement can be observed here.
Name 4 surrogate measurements of migration
- Video microscopy
- Assessing antagonists in whole blood
- Gated Auto-Fluorescence Forward Scatter (GAFS) - measures shape and granularity
- Caclium mobilisation
Explain how you can measure chemotaxis using video microscopy
Cells are placed on one side of a slide and chemoattractants on the other. The movement can be plotted and analysed on a vector plot
Explain how assessing antagonists in whole blood measures chemotaxis
- In vitro*: Blood is taken, and drug is given with stimulus. Shape change and CD11b expression measured using flow cytometry
- Ex vivo*: Drug is given, blood is then taken and then is stimulated. Flow cytometry is used to measure changes in shape and CD11b (integrin)
- CD11b mediates migration and adhesion amongst other things*
Explain how Gated Auto-Fluorescence Forward Scatter (GAFS) measures chemotaxis
Flow cytometry which measures shape with the forward scatter and granulartiy with the side scatter. Chemattractant can be used to induce shape change/granularity and analysed by fluorescent plotting.
Explain how Ca2+ mobilisation measures chemotaxis
Mobilisation activated by G protein-coupled receptors which increase intracellular Ca2+. Fluorescent dye will only bind to calcium when it is in the cytoplasm. Produces dose response curve with varying concentrations of chemokines
Uses signal transduction, surrogate to direct measurement of movement
Describe the dose-response curve observed with chemokine concentration and cell migration
Bell-shaped curve as supposed to sigmoid
Give a possible explanation to the bell-shaped curve seen in chemotaxis response curve. Describe the benefit of this response
Cell is polarised - it has a head and tail. When receptor binds to chemokne, receptors migrate to this side (the head) which is believed to lead the migration of the cell.
At high concentrations, the cell is surrounded by agonists and therefore there is no gradient. This means the cell does not know which way to move
Benefit: cell is attracted to fight off infection - it needs to be able to stop at site of injury where the concentration of chemoattractant will be very high
Chemokine-chemokine interactons could also pla a role in the bell-shaped curve observed in chemotaxis response curve.Explain how.
At high concentrations, chemokines can dimerise:
- Homodimers (CXCL8, CCL2, CCL5) - CXCL8 homodimers led to ↓CXCR1 binding - self-limiting to reduce chemotaxis
- Heterodimers (CXCL8-CXCL4) - ↑CXCL8/CXCR2-dependent migration and ↑CXCL4-dependent-anti-proliferative effect on EC
- Oligodimers: CCL17, CXCL4, CXCL7 etc
Neihbour et al, 2014, targeted chemokine receptor 3 (CCR3) to treat asthma. It was a double blind parallel group study where groups were given 300mg of antagonist or placebo for 10 days, then followed by 5 days of prednisolone. They induced sputum neutrophilia using ozone challenge but observed eosinophils.
They found:
- No effect on blood eosiophilia
- No effect FEV1
- Improvement in PC20 (provocative challenge to drop FEV by 20%)
- Improvement in Asthma Control Questionnaire (ACQ) score
Useless drug
In COPD, neutrophils are targeted through CXCR1/2 antagonists. They found that:
- Neutrophil migration through CXCL8 and CXCL1 acting on CXCR1/2.
- In COPD, greater response to CXCL1 compared to smokers and non-smokers
- PBMC chemotaxis CXCR1/2:
- ↑cells per feild with CXCL1 stimulus than CXCL8 (not seen)
- PBMC chemotaxis CXCR3:
- ↑chemotaxis from CXCL10, 9 and 11 compared to smokers and non-smokers
Traves et al Journal Leuk Biol 2004
The increased response of COPD neutrophls to CXCL1 is theorised to be due to receptor recycling. It is thought this process occurs mor rapidly in COPD leading to more response - reason is currently not known. Explain the concept of receptor recycling.
Migration is due to the recurrent binding of chemoattractant to its receptor. This is done through receptor recycling.
When ligand binds to receptor, G-receptor kinase (GRK) phosphorylates receptor internally by conformational change.
This recruits arrestin which is involved in endosomal trafficking.
The receptor can be destroyed in the endosome OR dephosphorylated and re-trafficked to the cell surface.
Compare the neutrophil migration of healthy and COPD patients and explain its effects on surrounding cells
Loss of direction in COPD
Move faster in COPD
Abberant migration of neutrophils cause more damage to surrounding cells and also inefficient pathogen clearance
Can be corrected by PI3K inhibitor as PI3K believed to be the molecular compass of the cell
Rennard et al, 2015, observed the effects of CXCR1/2 antagonists in treating COPD.
Double blind placebo controlled trial of two periods:
Period 1: 6 mths 10, 30, 50mg (n=460)
Period 2: 18 mths as above (n=275)
They found:
- ↓sputum neutrophils
- ↑FEV1 (67ml)
- ↑FEV1 smokers (168ml)
- ↓Absolute neutrophil count
Describe 5 ways in which we can reduced leukocyte recruitment (theoretically)?
- Inhibition of actin polymerisation/cytoskeleton
- Chemokine antibodies
- Chemkine receptor antagonists
- Dimerisation of receptors
- CCR5 ligands inhibit CCR2 ligand binding
- CCR5-CXCR4 modulate T cell function
- Dimerisation with delta opioid receptor
- CXCR4 homodimer = cellular effects
- Heterodmer = inhibition of these effects
