Lecture 2: Granulocytes Flashcards
What are granulocytes?
White blood cells with cytoplasms filled with granules containing antimicrobial/immunomodulatory proteins
What are the 2 stages of granulopoiesis?
Lineage Determination & Maturation
What is the process of granulopoiesis?
Producing and maturing granulocytes in the bone marrow, through differentiation of HSC into myeloblast precursor cells, followed by other stages to form fully mature granulocytes
What is the process of lineage determination, beginning with HSCs?
HSCs > common myeloid progenitors CMPs > granulocytes-monocyte progenitors GMPs stimulated by GM-CSF > multi-lobed granulocytes
What is the purpose of G-CSF?
Stimulates GMP, making myeloblasts, which differentiate into promyelocytes, then forming fully mature granulocytes
What are the 3 main transcription factors involved in lineage commitment of HSCs?
PU1, C/EBPs, and GATA-2
What does PU1 consist of?
- N-terminal transactivation domain
- PEST domain, serves as activation sequence to recruit transcriptional enhancer proteins
- conserved ETS domain at the C-terminus
How can PU1 be used to determine neutrophil maturity?
PU1 is highly detectable in the nucleus of neutrophil precursors, but absent during late maturation
What is the result of deleting PU1 gene?
Hematopoietic defects including loss of development of myeloid and lymphoid cells
What does PU1 do?
- Interacts with PU boxes in the genome to activate transcription of target genes
- Is expressed by myeloid and B lymphoid lineages, ETS domain mediated binding to DNA and interactions with other proteins
What are C/EBPs?
A family of 6 CCAAT-enhancer-binding proteins
Are transcription factors that bind DNA to open up chromatin which induces gene expression
What do C/EBPs consist of?
- Leucine zipper dimerisation domain at C terminus, to open sites of the genome for transcription
- A basic region
What is the result of C/EBP knockout?
Loss of mature neutrophils, decrease in monocyte frequency, differentiation block in transition from CMP to GMP
What cells are C/EBP epsilon found in?
Confined to granulocytic cells
What is GATA-2 useful for?
Crucial for proliferation and maintenance of HSCs and multipotent progenitors
What cells are GATA-1 expressed in?
Primitive and definitive erythroid cells, megakaryocytes, eosinophils, and mast cells
How does GATA cross regulatory mechanisms work in erythrocytes?
GATA-1 controls expression of GATA-2 (and vice versa)
High levels of GATA-2 activates GATA-1 and induces a feedback loop to prevent GATA-2 transcription
How many granule types are formed and in what order are they released ?
4 types, released backwards
Secretory > tertiary > secondary > primary
What is the bone marrow reserve?
A large storage pool of mature neutrophils in the bone marrow
What instigates mobilisation of neutrophils upon inflammation?
Chemokines and chemokine receptors
How is CXCL12 involved in retaining PMNs in the bone marrow?
- bone marrow expresses high levels of CXCL12 by stromal cells
- mature neutrophils express low levels of CXCR4 (ligand for CXCL12)
- allows less retention of mature neutrophils
How does GCSF regulate CXCR4 expression?
- GCSF reduces CXCR4, inducing mobilisation of neutrophils
- chemotaxis towards inflammatory site via chemoattractants CXCL1, CXCL2, IL8
- GCSF pushes granulopoiesis and downregulates CXCR4
- allows cells to migrate, then use CXCR2 to go to sites of infection
- neutrophil death increases CXCR4 expression for homing purposes
What are the stages of neutrophil extravasation?
Chemotaxis & rolling, adhesion, crawling, diapedesis, and migration
What initiates neutrophil rolling?
Changes on the endothelium surface resulting from inflammation (histamines, cytokines, leukotrienes)
What is the process of neutrophil rolling?
- endothelial cells become activated by PRRs, which unregulates pre-stored P-selectin
- E-selectin is produced
- P- and E- selectins bind to their glycosylated ligands (eg. PSGL-1)
- induces tethering onto the surface, and subsequent rolling in direction of blood flow
Where is P-selectin stored?
In Weibel Palade bodies
What causes chemotactic gradient in the intravascular space?
Chemokines are immobilised on the endothelium by binding negatively charged heparin sulphates
What do G coupled chemokine receptors do?
Induce signals for activation, resulting in expression of cell adhesion molecules (CAMs)
How do neutrophils have firm adhesion?
- neutrophils express LFA-1 and MAC1 constitutively at high levels
- these bind common endothelial cell CAMs produced by GCPRs
- LFA-1 and ICAM1 binding is essential for firm adhesion
