Leukocyte trafficking in rheumatoid arthritis Flashcards
what is the leukocyte adhesion cascade?
- how leukocytes exit blood and enter tissue
- Occurs as a protective inflammatory response to pathogens and damage
- Leukocytes roll along endothelium and migrate into tissue
how is the adhesion cascade implicated in disease?
This process underpins pathology
- In CVD, RA, liver disease and IBD – aberrant trafficking of leukocytes from blood into tissues – chronic inflammation in tissues
- Associated with ageing - changes with immune system and leukocyte recruitment profiles
what occurs during the adhesion cascade?
- Margination of leukocytes from centre of blood to vessel wall
- This happens in absence of inflammation
- Upon inflammation, endothelial cells begin to express receptors to mediate capture, rolling, firm adhesion and migration of leukocytes
- In resting state/healthy – endothelial cells act as barrier to keep blood and leukocytes within vessel
- Components within subendothelial space (basement membrane, fibroblasts and ECM) can influence endothelial phenotype and regulate this cascade
how is the recruitment cascade controlled?
- endothelial phenotype
- endothelial activation
- leukocyte activation once they have bound to the endothelial surface
what influences the endothelial phenotype?
Haemodynamic forces of flowing blood – this determines anatomical location where recruitment can occur:
- Shear stress within arteries are too high for leukocytes to form stable interactions with inflamed endothelium
- Leukocyte recruitment tends to occur in postcapillary venules and at vessel bifurcations (link to CVD)
Stromal microenvironment e.g. ECM, fibroblasts
- cytokines derived from leukocytes e.g. macrophages, or damaged cells (DAMPs)
Hypoxia
what influences endothelial activation?
- Endothelial response to cytokines, inflammatory mediators, endotoxin, hypoxia/reperfusion, damage induced by hypoxia
- Upon activation, endothelial cells modify expression of adhesion receptors
what happens when the endothelium is activated?
It upregulates capture and adhesion receptors
- Endothelial cells present chemokines and lipids
- Anything released from endothelium into blood will be washed away, so molecules need to be presented on surface to interact with leukocytes
what influences leukocyte activation and what does this induce?
- become activated once bound to endothelial surface
- Response to activating stimuli (cytokines, chemokines, lipids)
- Integrin activation of leukocytes – changes to a high affinity state to stabilise binding
- Cytoskeletal rearrangement of leukocyte – spherical to flattened on endothelial surface to reduce frictional forces from flowing blood
how does the phenotype of leukocytes change?
Shape change via cytoskeleton
Metabolism of T cells can affect how migratory they are
what is margination?
Margination: red blood cells form route down centre of blood vessel
- they become the largest item within flowing blood
- This pushes platelets and leukocytes towards the vessel wall
– allows leukocytes and platelets to contact vessel wall
- Under inflammation, leukocytes are now close enough to interact with receptors on endothelium
why do leukocytes roll along the vessel wall?
Endothelial activation causes upregulation of capture receptors e.g selectins
- Density of selectins dictates speed of cell rolling
- High density = slower rolling, less dense = faster rolling
- Intermittent density = stop-go interactions, cell binds selectin and bounces off over and over again
- Rolling occurs because kinetics of selectin interaction with receptors have fast on and off rates
- The interaction isn’t weak, but the dynamics of binding are rapid
- Physical forces of blood flow cause interactions to form and dissociate quickly, enabling cells to roll to next interaction
how are neutrophils captured?
Neutrophils are captured from flow by selectin coated surfaces, not ICAM-1 coated surfaces
- ICAM-1 is an immunoglobulin superfamily members
- If you coat glass coverslips with ICAM and perfuse over neutrophils, there is no capture from flow
- If neutrophils are perfused over a surface coated with CD62, there is capture from flow
- There is decrease in capture as wall shear stress increases
what is the nomenclature for selectins?
- CD62 is the CD number for selectins
P-selectin = CD62p, same for E and L
how are T cells captured?
Occurs under TNFa+IFNy-induced inflammation:
- T cells can be captured from flow mainly through E-selectin on endothelial cells
- They can also be captured via a4b1 integrins – acts as capture receptor of T cells, which binds VCAM on the endothelium
how can T cell capture be studied?
by inhibiting capture receptors on the endothelium
by inhibiting capture receptors on the lymphocyte
what happens to T cell capture when endothelial capture receptors are blocked?
T cell binding to endothelial cells in response to inflammation – antibodies added to block endothelial cell receptors
- In absence of antibody, there is 100% binding (normalised to no antibody control)
- Blockage of VCAM domain 4, or 1 and 4 = there is inhibition of T cell binding
- Blockage of E-sel in conjunction with VCAM inhibition also impairs T cell binding
- Both VCAM and E-sel are required for T cell capture
what happens to T cell capture when lymphocyte capture receptors are blocked?
Antibodies blocking lymphocyte adhesion receptors:
- Treat with a4 integrin antibody = reduced T cell binding
- Reduction not seen when b2 integrin is blocked
a4 bound to b1 integrin subunit is the ligand for VCAM
why don’t leukocytes continue to roll?
due to endothelial activation
- upregulation of IgSF adhesion receptors - density and strength
- presentation of chemokines and lipids e.g. PGD2
due to leukocyte activation
- high affinity integrin activation
- cytoskeletal rearrangement
how do endothelial cells become activated to stop leukocyte rolling for stabilisation?
Endothelium not only upregulates selectin capture receptors, but also upregulates adhesion molecules – IgG superfamily members ICAM and VCAM
- ICAM and VCAM form bonds and detach bonds slowly with their ligands – slow kinetics
– not stronger interactions, but slower dynamics
- As kinetics are slower, cells can no longer roll
- Endothelial cells also express chemokines and lipids
- Chemokines signal to the leukocyte to induce integrin activation
how do leukocytes become activated to stop rolling for stabilisation?
Integrin activation of leukocytes changes their affinity state
- Low affinity to high affinity state
- There is activation of the leukocyte, causing cytoskeletal rearrangement, becoming flattened against endothelium
how do neutrophils become stabilised during the adhesion cascade?
CXCR2-chemokine induces b2-integrin activation to support stable adhesion
- CXCR2 on the neutrophil binds CXCL1 and CXCL5 on the endothelium
- that neutrophils receive signal via CXCR2 – this causes activation of b2 integrins, changing affinity state to stabilise adhesion
how can neutrophil stabilisation be studied?
- Blocking CXCR2 = reduction in neutrophil binding, measured by reduction in neutrophil transmigration - inhibits stability of neutrophil adhesion, leading to rolling
- Blocking b2 integrins on neutrophils reduces adhesion
what are the two chemokine receptors typically expressed on neutrophils?
2 chemokine receptors on neutrophils:
- CXCR1 which binds IL-8 (CXCL)
- CXCR2 which binds CXCL1 and CXCL5
what is chemokine inside-out signalling?
Integrins on leukocyte are inactive until they encounter chemokines expressed on surface of endothelium
- When chemokine binds leukocyte chemokine receptor, it upregulates integrin expression to then bind ICAM and VCAM on endothelium, enabling flattening
- Endothelium chemokines activate migration of leukocytes – stabilised adhesion and cytoskeletal changes
how do T cells become stabilised during the adhesion cascade?
T cell stabilisation requires interaction with CXCR3
- This causes a4b1 integrin to become activated, leading to firm adhesion via VCAM1
- Also requires prostaglandin signal via DP2 receptor to support onward migration of adherent T cells along endothelium
how can T cell stabilisation be studied?
CXCR3 blocking antibody reduces T cell adhesion
Inhibition of DP2 receptor (receptor for prostaglandin D2) blocks subsequent transendothelial migration of T cells
what adhesion molecules are important for leukocyte migration?
- ICAM1 and VCAM1 (Ig superfamily members)
- Junctional molecules e.g. CD31 and JAMs (junctional adhesion molecules)
- Lipids e.g. prostaglandin D2
what are the main stages of leukocyte migration?
- Migration across endothelial surface (abluminally)
- Transendothelial
- Subluminal/subendothelial migration
- Trans-basement membrane
Once cells have penetrated basement membrane, they are now within tissue and have left vascular barrier
what controls neutrophil migration?
- Need signal via DP1 receptor for PGD2
- Need signal via b2 integrins for across and through endothelial
- Need signal via b1 integrins for migration through and beneath endothelium
how can neutrophil migration be studied?
Inhibitor to DP1 receptor = reduction in neutrophil migration
Block b2 integrin = reduction in neutrophil transendothelial migration
Block b1 integrin = reduction in neutrophil subendothelial migration
how do neutrophils and T cells differ in their prostaglandin receptors?
DP1 receptor on neutrophils is different to the DP2 receptor that T cells require to migrate
- But both mediate PGD2 signal
how are prostaglandins generated?
Prostaglandins generated by arachidonic acid via COX1 and 2 from endothelium and leukocytes
- When in tissue, leukocytes become activated by PAMPs/DAMPs
what controls T cell migration?
T cell migration mediated by ICAM1 and b2 integrins
- In addition to prostaglandin, integrin and ICAM signals, increase in sphingosine-1-phosphate (S1P) dose causes inhibition of T cell migration across endothelial cells
- T cell migration can be negatively regulated by different lipids
how can T cell migration be studied?
Blockage of ICAM1 or LFA-1 (alphaL-b2 integrin/CD11a/CD18) reduces T cell migration
- blocking of VCAM1 has no effect
what is leukocyte transendothelial migration?
migration through the endothelium
- complex
- involves junctional molecules, basement membrane
what junctional molecules control migration?
Junctional molecules control paracellular migration – ICAM-2, JAM-A, and PECAM-1 (CD31) act sequentially to control neutrophil migration across postcapillary venules into inflamed tissue in vivo
- Paracellular = migration of leukocyte through junctions
what must leukocytes do once they have migrated through the endothelium?
Once through endothelium, leukocytes have to traverse the basement membrane
- Not much is known about the molecules involved in T cell migration across basement membrane, so this data is mostly on neutrophils
- Basement membrane has regions of low expression of proteins
- These are mirrored in the gaps in the pericyte coverage and basement membrane
- In the gap is a neutrophil
- These low expression regions in the basement membrane are where leukocytes preferentially migrate through
what markers show gaps in the basement membrane?
LNa5 = laminin
A-SMA = smooth muscle actin
CD11b = neutrophil marker
what proteins do neutrophils use to migrate through the gaps of the basement membrane?
Neutrophils use integrins in a series:
- b2 integrins to migrate through endothelium
- b1-integrins for migration through and underneath endothelium
- They start using b1 integin to start migrating through basement membrane
- b3-integrins for migration across the basement membrane – take over from b1
