Chemokines & Recruitment of Leukocytes Flashcards
What are chemokines?
- Induce chemotaxis
- Promote leukocyte recruitment during development, homeostasis and inflammation
- Nomenclature based on number of amino acids between two cysteines, eg) CC, CXC…
- All chemokines related in amino acid sequence
- Their receptors are GPCRs
- Signalling pathway initiated by chemokines causes changes in cell adhesiveness and the cytoskeleton that leads to directed migration.
- Can be secreted by many cells, not just immune cells
- In the immune system, function mainly to recruit effector cells of innate immunity from the blood into sites of infection.
- CC chemokines → recruitment of monocytes and lymphocytes. Eg, CCL2 attracts monocytes.
- CXC chemokines → recruitment of neutrophils. Eg, CXCL8
Describe the role of selectins, ICAMs and integrins.
Selectins:
• Membrane glycoproteins with a distal lectin-like domain that binds specific carbohydrate groups
• induced on activated endothelium
• Bind to fucosylated oligosaccharide ligands on passing leukocytes (Sialyl-Lewis proteins)
• Cytokines, particularly TNFa, induce externalization of Weibel-Palade bodies:
− Contain preformed P-selectin → appears on the surface of the endothelium just after macrophages have responded to the presence of microbes
− Shortly after, mRNA encoding E-selectin is synthesised
ICAMs & Integrins:
• The next step in leukocyte recruitment depends on tighter adhesion, due to binding of intercellular adhesion molecules (ICAMs) on the endothelium to heterodimeric proteins of the integrin family on leukocytes.
• ICAMs are single-pass membrane proteins belonging to the Ig-like superfamily
• The integrins important for extravasation are LFA-1 and CR3
• LFA-1 and CR3 bind to ICAM-1 and ICAM-2 → even in the absence of infection, circulating monocytes are continuously leaving the blood to become resident macrophages. Here, they use ICAM-2, which is expressed at low levels by unactivated endothelium
• Strong adhesion is promoted by the induction of ICAM-1 on inflamed endothelium, together with a conformational change in LFA-1 and CR3 that occurs on the leukocyte (integrins can switch between an active and inactive state)
− In the activated state, an integrin is linked via talin to the actin cytoskeleton
− Chemokines binding to the receptors on leukocytes cause talin to bind to the cytoplasmic tails of the beta chains of LFA-1 and CR3, so they assume the active conformation.
Describe step 1 of leukocyte extravastation.
Step 1 – Tethering:
• Circulating neutrophils tether to the endothelium
• Loose attachment – can bind and unbind – will unbind if there is no injury/nfection stimulus
• Mediated by P, E and L selectin:
− L selectin is expressed by most leukocytes
− P & E selectin expressed by inflamed endothelial cells
• Cytokines, particularly TNFa, induce externalization of Weibel-Palade bodies:
− Contain preformed P-selectin → appears on the surface of the endothelium just after exposure to leukotriene B4, C5a, histamine or TNFa
− Shortly after, mRNA encoding E-selectin is synthesised
• PSGL1 has a dominant role as a ligand for all selectins, mediates leukocyte-endothelial attachment or leukocyte-leukocyte interactions (so adherent leukocytes facilitate secondary leukocyte capture).
Describe step 2 of leukocyte extravasation.
Step 2 – Rolling:
• Interaction of P and E selectin with PSGL1 allows leukocytes to adhere reversibly to the vessel wall, so they can be seen to roll along the endothelium
• Requires shear stress to support adhesion – rolling cells detach if blood flow is stopped.
Describe step 3 of leukocyte extravasation.
Step 3 – Arrest:
• Depends on interactions between leukocyte LFA-1 and CR3 with endothelial ICAM-1 (induced by TNFa) and ICAM-2.
• LFA-1 and CR3 normally bind their ligands weakly, but CXCL8 binds to chemokine receptors on the leukpcyte and signals the cell to trigger the conformational change in LFA-1 and CR3 that increase the adhesive properties:
− In the activated state, an integrin is linked via talin to the actin cytoskeleton
− Chemokines binding to the receptors on leukocytes cause talin to bind to the cytoplasmic tails of the beta chains of LFA-1 and CR3, so they assume the active conformation → results in opening of the ligand binding pocket.
− Known as inside-out signalling
Describe step 4 of leukocyte extravasation.
Step 4 – Adhesion strengthening:
• Involves the stabilization of adhesion and full neutrophil activation
• Ligand-induced integrin clustering contribute to the initiation of outside-in signaling and the formation of signalosomes, required for recruitment of SRC kinases
• The SRC kinases FGR and HCK are not required for inside-out signaling and adoption of the active binding conformation, however lack of outside-in signaling greatly accelerates the detachment of adherent neutrophils under flow.
• The SRC kinases function to change cell motility and spreading.
Describe step 5 of leukocyte extravastion.
Step 5 – Crawling:
• Prior to crossing the endothelium, neutrophils ‘crawl’ to find the preferred sites of transmigration.
• Dependent on the neutrophil integrin Mac-1 interacting with endothelial ICAM-1.
• When crawling is disabled, transmigration is delayed and occurs through the transcellular pathway as opposed to paracellular.
• Adherant leukocytes induce the formation of ‘docking structures’ or ‘transmigratory cups’ - endothelial cell projections rich in ICAM and VCAM, which may initiate transmigration.
Describe step 6 of leukocyte extravasation.
Step 6 – Transendothelial cell migration
• Involves LFA-1 and CR3, as well as a further adhesive interaction involveing an Ig-related molecule called PECAM expressed on the leukocyte and at the intercellular junctions of endothelial cells.
• Paracellular route:
− Through endothelial junctions
− Ligation of endothelial cell adhesion molecules may lead to reduced inter-endothelial contact and facilitate leukocyte migration through junctions.
− Also dependent on increased levels of intracellular endothelial Ca2+ → promotes transmigration through the opening of endothelial cell contacts via activation of MLCK and subsequent endothelial cell contraction.
− Inflamed endothelial cells can re-distribute junctional molecules that serve as obstacles such as E-cadherin than, and junctional molecules for which leukocytes express ligands (eg, PECAM) can mobilise to the junction.
• Transcellular route:
− Thought transmigration only occurred via the paracellular pathway, but transcellular migration occurs in the CNS.
− Occurs for only 5-20% migrating cells
• Penetrating the endothelial cell barrier can be rapid,
Describe step 7 of leukocyte extravasation.
Step 7 – migration through the basement membrane
• Final step is migration through the tissues under the influence of chemokines – chemotaxis
• CXCL8 and CCL2 produced at the site of infection/injury and bind to glycosaminoglycans in the ECM or endothelial surface creating a matrix-associated chemokine gradient
• CXCL8 is released by the macrophages first encountering the challenge – so neutrophils are the first to be recruited. Their influx peaks within the first 6 hours, after which monocytes are recruited.
• The endothelial BM is composed of laminins and collagen IV.
− There are regions of the BM that have lower than average BM constituents – more permissive to emigrating neutrophils
− IL-1B results in transient enlargement of these sites
− These sites are co-localised with gaps between pericytes, so neutrophil migration occurs specifically at regions of least resistance.
What is the role of neutrophils in brain injury?
• Stroke induces CXC chemokine production
McColl et al,:
• Systemic inflammation (eg, infection) increases the risk of stroke and associated with poorer clinical outcome
• Brain damage and neurological deficit 24h after MCA occlusion exacerbated by systemic LPS administration.
• Exacerbaton dependent on IL-1, because co-admin of IL-1RA abolished the effect of LPS on brain damage
• Systemic admin of IL-1 increased ischemic damage to a similar extent as LPS, increased levels of IL-6 and acute phase proteins, and increased neutrophil selective chemokines.
• Neutrophil mobolisation were aggravated by IL-1 before increased ischaemic damage seen.
• Neutropenia abolished effects of systemic IL-1
➢ Suggests that IL-1 induced potentiation of neutrophil mobilization via CXC chemokines is detrimental to outcome after stroke
➢ May explain poorer outcome in stroke patients presenting with infection.
→ Chemokine production is exacerbated by systemic inflammation (show in the study by injection of LPS or IL-1)
→ Inflammatory mediators synergise with the injury to increase chemokines
→ Stroke increases circulating neutrophils and neutrophil recruitment.
How important are these neutrophils to injury?
• Can target the things neutrophils produce, eg, MMPs → this is protective
• Deplete neutrophils from an organism using PMN antibody → reduces the brain injury induced by stroke
• Neutrophils are looking
