Lecture 2: Granulocytes

Question 1

What are granulocytes?

Answer 1

White blood cells with cytoplasms filled with granules containing antimicrobial/immunomodulatory proteins

Question 2

What are the 2 stages of granulopoiesis?

Answer 2

Lineage Determination & Maturation

Question 3

What is the process of granulopoiesis?

Answer 3

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

Question 4

What is the process of lineage determination, beginning with HSCs?

Answer 4

HSCs > common myeloid progenitors CMPs > granulocytes-monocyte progenitors GMPs stimulated by GM-CSF > multi-lobed granulocytes

Question 5

What is the purpose of G-CSF?

Answer 5

Stimulates GMP, making myeloblasts, which differentiate into promyelocytes, then forming fully mature granulocytes

Question 6

What are the 3 main transcription factors involved in lineage commitment of HSCs?

Answer 6

PU1, C/EBPs, and GATA-2

Question 7

What does PU1 consist of?

Answer 7

• N-terminal transactivation domain
• PEST domain, serves as activation sequence to recruit transcriptional enhancer proteins
• conserved ETS domain at the C-terminus

Question 8

How can PU1 be used to determine neutrophil maturity?

Answer 8

PU1 is highly detectable in the nucleus of neutrophil precursors, but absent during late maturation

Question 9

What is the result of deleting PU1 gene?

Answer 9

Hematopoietic defects including loss of development of myeloid and lymphoid cells

Question 10

What does PU1 do?

Answer 10

• 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

Question 11

What are C/EBPs?

Answer 11

A family of 6 CCAAT-enhancer-binding proteins

Are transcription factors that bind DNA to open up chromatin which induces gene expression

Question 12

What do C/EBPs consist of?

Answer 12

• Leucine zipper dimerisation domain at C terminus, to open sites of the genome for transcription
• A basic region

Question 13

What is the result of C/EBP knockout?

Answer 13

Loss of mature neutrophils, decrease in monocyte frequency, differentiation block in transition from CMP to GMP

Question 14

What cells are C/EBP epsilon found in?

Answer 14

Confined to granulocytic cells

Question 15

What is GATA-2 useful for?

Answer 15

Crucial for proliferation and maintenance of HSCs and multipotent progenitors

Question 16

What cells are GATA-1 expressed in?

Answer 16

Primitive and definitive erythroid cells, megakaryocytes, eosinophils, and mast cells

Question 17

How does GATA cross regulatory mechanisms work in erythrocytes?

Answer 17

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

Question 18

How many granule types are formed and in what order are they released ?

Answer 18

4 types, released backwards

Secretory > tertiary > secondary > primary

Question 19

What is the bone marrow reserve?

Answer 19

A large storage pool of mature neutrophils in the bone marrow

Question 20

What instigates mobilisation of neutrophils upon inflammation?

Answer 20

Chemokines and chemokine receptors

Question 21

How is CXCL12 involved in retaining PMNs in the bone marrow?

Answer 21

• 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

Question 22

How does GCSF regulate CXCR4 expression?

Answer 22

• 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

Question 23

What are the stages of neutrophil extravasation?

Answer 23

Chemotaxis & rolling, adhesion, crawling, diapedesis, and migration

Question 24

What initiates neutrophil rolling?

Answer 24

Changes on the endothelium surface resulting from inflammation (histamines, cytokines, leukotrienes)

Question 25

What is the process of neutrophil rolling?

Answer 25

• 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

Question 26

Where is P-selectin stored?

Answer 26

In Weibel Palade bodies

Question 27

What causes chemotactic gradient in the intravascular space?

Answer 27

Chemokines are immobilised on the endothelium by binding negatively charged heparin sulphates

Question 28

What do G coupled chemokine receptors do?

Answer 28

Induce signals for activation, resulting in expression of cell adhesion molecules (CAMs)

Question 29

How do neutrophils have firm adhesion?

Answer 29

• 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