Innate Cells (Granulocytes, NK Cells) Flashcards
Granulocytes, NK Cells
Define a granulocyte and give examples.
a type of white blood cell with granule-filled cytoplasm. These granules contain proteins that often have antimicrobial or immunomodulatory functions.
Granulocytes are often the first cells to be recruited to a site of infection
- neutrophil
- eosinophil
- basophil
These originate from common myeloid progenitors
NK cells and monocytes have granules, but are far less abundant and thus considered agranulocytes
What is granulopoiesis?
The process of producing and maturing granulocytes in the bone marrow
differentiation of hematopoietic stem cells into precursor cells called myeloblasts followed by several stages of maturation
stage 1. lineage determination
stage 2: maturation
What is the structure of PU.1?
A TF that contains:
- N terminal transactivation domain
- a PEST domain which serves as an activation sequence to recruit transcriptional enhancer proteins eg PIP
- a conserved ETS domain -> binds DNA
PU.1 interact w PU boxes in genome to activate transcription of genes
What is the function of PU.1?
development of lymphoid stem cells from HSCs requires low levels of PU.1
development of myeloid stem cells from HSCs requires high levels of PU.1
– stage of maturation of a granulocyte can be
determined by PU.1 levels, high = immature
deletion of PU.1 results in loss of development of myeloid and lymphoid cells
can also regulate GATA-1 transcription, inhibiting erythroid cell fate
GATA-1 can inhibit PU.1 to inhibit myeloid cell fate
What is the structure and function of C/EBPs?
a family of 6 CCAAT-enhancer binding proteins
TFs that bind DNA to open chromatin and induce gene expression
– C terminal leucine zipper dimerisation domain (LZ)
– basic region (BR)
Where are different C/EBPs found?
- gamma and beta found in nearly all cells
- epsilon is largely confined to granulocytic cells
play essential roles in the development, differentiation
loss of alpha in BM = complete loss of mature neutrophils and decrease in monocytes
– differentiation block from common myeloid progenitor (CMP) into granulocyte-monocyte progenitor (GMP)
What is the function of the GATA TFs?
GATA-1: expressed in primitive and definitive erythroid cells, megakaryocytes, eosinophils, and mast cells
- GATA-1 negative = neutrophil
- GATA-1 positive = eosinophil
GATA-2: crucial for proliferation and maintenance of HSCs and multipotential progenitors
– in erythrocytes, cross-regulatory mechanism by which GATA-1 can control expression of GATA-2 and vice versa
GATA-3: master regulator of Th2 differentiation and function
Describe the function of neutrophils.
large pool of mature neutrophils in BM, “bone marrow reserve”
make up 50-70% of all WBCs in the blood
rapidly mobilised during inflammation
egress from BM can increase circulating neutrophil numbers 10-fold, in hours
contain many antimicrobial granular proteins which are released through NETosis
How are neutrophils mobilised?
high levels of CXCL12 in the BM
neutrophils express low levels of CXCR4 (CXCL12 ligand)
– this axis keeps mature (polymorphonuclear) neutrophils in BM
G-CSF reduces CXCR4, thus inducing mobilisation
chemotactic signals from inflammatory site allow specific location targeting eg IL-8, CXCL1/2, C5a
OUTSIDE READING YAAAY
- circulating neutrophils found to express increasing levels of CXCR4 independent of inflammatory markers (in vitro)
- at certain age blood neutrophils home to BM to be phagocyted by resident macrophages, which causes release of G-CSF (positive feedback loop)
- neutrophils in tissue phagocytosed by tissue resident macrophages
– MACROPHAGES EXPRESS CXCL12
What are the steps of neutrophil extravasation?
- rolling
- adhesion
- crawling
- transmigration
outline rolling.
- when endothelial cells are activated (eg by PRRs) they upregulated stored P-selectin from Weibel-Palade bodies
- E-selectins (stronger) produced de novo within 90 minutes
- P and E-selectin bind to selectins on neutrophil such as P-selectin glycoprotein 1 (PSGL-1)
- cytokines can increase selectin expression on endothelial cells eg TNFa = increased e-selectin
- this processes induces tethering, and subsequent rolling in direction of blood flow
outline adhesion.
- chemokines (basic) are immobilised on the endothelium by binding heparan sulphates (a GAG) (negative) -> chemotactic gradient in the intravascular space that guides neutrophils
- G-protein-coupled receptors (GPCRs) on neutrophils (eg CXCL1/2/8) bind chemokines
- neutrophils express LFA-1 and MAC1 constitutively at high levels in a bent conformation that cannot bind to CAMS.
Binding of chemokines to GPCRs activates these receptors allowing them to interact w ICAM1 which is essential for firm adhesion
outline crawling and tissue migration.
once in the right location, neutrophils need to crawl to find their preferred site of transmigration
this is dependent on MAC-1 and ICAM1
- ligation of ICAM1 is associated w increased intracellular Ca2+ and activation of p38 mitogen-activated protein kinase (MAPK) and RAS homologue (RHO) GTPase
- These events promote paracellular migration
– leukocytes can also migrate through the endothelium (transmigration)
– transmigration induces cell-surface expression of beta1-integrins and proteases on neutrophils to aid migration eg MMPs, neutrophil elastase (NE)
How can our knowledge of extravasation aid in treatment of inflammatory conditions?
blocking VLA4 (natalizumab) in MS, and LFA1 (efalizumab) in severe psoriasis -> modulate or inhibit tissue inflammation
Give some examples of neutrophil activators?
- LPS
- TNF
- Chemokines (eg IL-8)
- growth factors
- PRRs eg dectin-1, a c-type lectin that binds fungal beta-glucan (most abundant PRR on neutrophils)
- opsonic receptors (complement receptors, Fc receptors)
fMLP (formyl peptide): a bacterial product that signals via a GPCR
- induces phagocytosis, chemotaxis and cell adhesion
- activates MAPK, PI3K, and NF-kB pathways
- comes from degraded bacterial or mitochondrial pathways (PAMP/DAMP)
How does a cell degranulate?
granules translocate to the phagosomal or plasma membrane to dock and fuse to release their contents
exocytosis of granules requires, at least, an increase in intracellular calcium (PMA(?))
What is the function of the granule, lactoferrin?
chelates two ferric ions (Fe3+)
- sequesters iron and prevents pathogen growth
- enhances NK cell activity
- can inhibit NETosis, by producing a shell around the cell
upregulated w inflammation -> biomarker of disease
What is the function of neutrophil elastase?
a major serine protease in primary granules, released in localised areas or attached to DNA in NETs
cleaves neutral, non-aromantic dipeptides -> broad array of substrates
degraded ECM, important for cell migration but also induces tissue injury
- immunotherapy target
digest microbes eg e coli OMP A
What is the function of MMP-9?
matrix metallopeptidase 9, a type IV collagenase which degrades ECM
also produced by macrophages and fibroblasts for tissue remodelling
also regulates migration/infiltration of neutrophils
excessive MMP-9 is found in rheumatoid arthritis and cardiovascular diseases contributing to tissue destruction
What is the function of MPO?
myeloperoxidase, found in neutrophils and monocytes
catalyses production of ROS; using hydrogen peroxide and chloride ions to generate hypochlorous acid (HOCl), a potent antimicrobial
– HOCl contributes to oxidative burst
levels of MPO in serum can indicate innate immune activation
What is NETosis?
a form of cell death, which utilises a web like structure composed of DNA and proteins that contains granules with AMPs
excessive release can cause tissue damage
What are azurophilic granules?
also known as primary granules
made first, largest granules
function is microbial killing inside phagolysosomes
eg MPO
What are specific granules?
also known as secondary granules
made second
function is to further aid in microbial killing
eg lactoferrin, NGAL (also sequesters iron), NADPH components (necessary for respiratory burst)
What are gelatinase granules?
also known as tertiary granules
made third
primarily involved in neutrophil migration through tissues eg MMP-9
