Term 2 Lecture 5: Haemopoiesis & Blood Components

Question 1

Humoural immunity

Answer 1

Soluble compounds of the immune system

Cytokines- A low molecular weight, secreted protein that stimulate/inhibit cell differentiation/proliferation

Interleukins - A group of cytokines that enable communication between leukocytes and particularly lymphocytes

Chemokines - structurally related substances that induce chemotaxis and activation of leukocytes

Question 2

Haemopoiesis: blood cell formation

Answer 2

Haeme = blood
Poeisis = formation

• yolk sac is the primary site during embryological development
• the bone marrow is the major site of blood cell production (haemopoiesis) by 20th week of gestation, increased activity in third trimester of pregnancy
• at birth haematopoietic cells (red marrow) occupy all bone marrow space
• in adults bone marrow stem cells are used for haematopoiesis - regulated by cytokines and growth factors

Question 3

Haemopoiesis timeline conception to birth

Answer 3

First trimester: yolk sac haemopoiesis (primitive wave) yields nucleated RBCs. Haemopoiesis is extraembryonic occuring in blood islands of yolk sac

Second trimester: HSCs then seem to migrate via blood stream to liver and spleen to seed these tissues, which then carry the burden of hemopoiesis during second trimester

7 months onwards: haemopoiesis in bone marrow

Primitive wave is prehepatic phase

Definitive wave is hematosplenothymic and medullolymphatic phase

Question 4

Bone marrow

Answer 4

Red: contains stem cells involved in hematopoiesis - present in all bones in a child and in the ribs, cranium, vertebrae, sternum,pelvis and ends of long bones in adults (absent in arms and legs after shoulder/hip joint)

Yellow: contains adipose tissue and is inert, during childhood gradual replacement of red marrow w/yellow fatty marrow occurs

When there is continuous increased hematopoietic demand yellow marrow throughout the body may become red and active again. The spleen and liver are also capable of producing blood cells and are the main site of extramedullary haematopoiesis

Question 5

Bone marrow, human evolution etc.

Answer 5

HSCs occupy a well protected niche
Bone marrow is a high calorie source providing fuel for expensive organs like the brain. Use of bone marrow fat may have allowed for enlargement of the brain.
Blood stem cells are protected from sunlight irradiation damage by being stored in bone marrow.

NOTE: bearded vultures eat only bone marrow and are not particularly smart so it is not as simple as eating bone marrow = intelligence

Question 6

Protection from UV is an evolutionarily conserved feature of the haematopoietic niche

Answer 6

Melanocytes protect stem cells present in the kidneys of fish such as in zebra fish. This is viewable by labelling kidney tubule epithelia with green marker protein and labelling stem cells with red markers. In wild-type fish the melanocytes cover the stem cells whereas in mutants w/out melanocytes stem cells remain visible.

Terrestrial animals have their stem cells in bone marrow for increased UV protection

Question 7

Stem cells

Answer 7

Self renew, differentiate into a range of lineages, slow replication

Types:
Totipotent - stem cell can develop into any tissue type, present in embryo/extraembryonic zone
Pluripotent - can become any of the three dermis layers - endo/ecto/mesoderm
Multipotent - can develop into only a specific lineage (can be manipulated in lab to become pluripotent)

Question 8

Stem cells to T & B cells

Answer 8

Hematopoietic stem cell
Converts to lymphoid stem cell
Then to T or B precursor
To T cell/ T killer cell or B cell
B cell can further develop into a plasma cell

Question 9

Stem cell to macrophage/neutrophil

Answer 9

Haematopoietic stem cell
Myeloid stem cell
Granulated macrophage CFU
Then: monoblast>promonocyte>monocyte> macrophage
Or
Myeloblast > promyelocyte > myelocyte>metamyelocyte>neutrophil

Question 10

Stem cell to eosinophil, basophil or mast cell

Answer 10

Hematopoietic stem cell
Myeloid stem cell
Eosinophil CFU > myeloblast> promyelocyte > myelocyte>metamyelocyte> eosinophil
Or
Basophil CFU>”>”>”>”> Basophil
Or just before becoming basophil become mast precursor and then mast cell

Question 11

Stem cell to platelets

Answer 11

Haematopoietic stem cell
Myeloid stem cell
Megakaryocyte CFU>megakaryoblast>megakaryocyte>platelets

Question 12

Stem cell to RBC

Answer 12

Haematopoietic stem cell
Myeloid stem cell
Erythroid CFU > primitive/mature progenitor (react to EPO) > pro erythrocyte>basophilic erythroblast >polychromatiphilis erythroblast > orthochromatic erythroblast (stage where RBC loses it’s nucleus) > reticulocyte>erythrocyte (RBC)

Question 13

Terminology for blood cell genesis from stem cells

Answer 13

Erythropoiesis - RBC genesis
Thrombopoiesis - platelet genesis
Granulopoiesis - neutrophil/basophil/eosinophil genesis
Lymphopoiesis - lymphocyte genesis
Monopoiesis - monocyte genesis

Question 14

Erythroid lineage

Answer 14

Erythrocytes (RBCs) most abundant cell type in the blood

Contain haemoglobin (alpha2 and beta 2 chains in adults)

Contain no typical organelles or cytomembranes in cytoplasm IN HUMANS - reptiles amphibians and birds have nuclei in their RBCs

Average lifespan 120 days, senescent RBCs phagocytosed by macrophages in liver and spleen

Lack of O2 (hypoxia) or decrease of erythrocytes to circulating blood (anaemia) caused by excessive destruction of RBCs, bleeding, iron or B12 deficiency leads to stimulation of interstitial cells in renal cortex to synth and release glycoprotein erythroprotein (EPO) into the blood

EPO stimulates early stages of erythroid colony forming unit (CFU) to proliferate and differentiate to basophilic > polychromatiphilic > orthochromatic erythroblast.

The pro erythroblast is the first stage of RBC lineage and derives from mature progenitor (w/ nucleus and free ribosomes for Hb synth) on stimulation of EPO.
Synth of Hb proceeds and as it accumulated the nucleus is reduced in size. Chromatin condenses, free ribosomes decrease cell shrinks and nucleus of cell migrated to outer membrane where it is ejected. The cell then becomes a reticulocyte which finally matures to an RBC. The nuclei are taken up by macrophages.

Question 15

Reticulocytes

Answer 15

Have no nuclei, methylene blue stain shows a network of strands in the cytoplasm (RNA), these cells are slightly larger than erythrocytes

Question 16

Erythroblastic island

Answer 16

Most important structural element of erythropoiesis
Found in bone marrow
Composed of a central macrophage surrounded by erythrocyte precursors

Macrophage complexes iron with ferritin and transfers this to the reticulocytes where it becomes Hb

Question 17

Red bone marrow

Answer 17

Consists of RBCs at diff stages of development and supporting tissue stroma rich in collagen and fibroblasts. Stem cells of white and red BCs & megakaryocytes divide to form platelets. Blood vessels running through the marrow are 1 epithelial cell thick through which mature RBCs can squeeze into the vessel

Question 18

Large cells need a large nucles

Answer 18

Or multiple nuclei. A large cell cannot function with a small nucleuso

Question 19

Control of thrombopoiesis

Answer 19

Thrombopoeitin (TPO) controls thrombopoiesis - platelet production

Megakaryocytes have TPO receptors on cell surface membrane as do the platelets as they are formed from this membrane. TPO is made in the liver and platelets bind to it in the blood system, sequester and inactivate it. So v. little TPO reaches the bone marrow and less megakaryocytes are converted to platelets. If there are not enough platelets less TPO is bound and so more reaches the bone marrow and more megakaryocytes are converted to platelets.

Question 20

Control of erythropoeisis

Answer 20

Erythropoietin EPO controls erythropoiesis - RBC production

EPO is made by kidney cells primarily and also in the liver. Release is triggered by reduced oxygen levels (hypoxia)

Erythropoeisis is also affected by:
Interleukins (IL-1, IL-3, IL-6)
Protein (SF-E - colony stimulating factor)
B12 and folic acid for maturation
Fe, Cu, Zn,Co & vit c for Hb synth

Question 21

More on erythroprotein EPO

Answer 21

34kd (v. small) glycoprotein produced mainly in kidney peritubular cells

Liver is secondary source

165 aa long and acts as a hormone

Glycosylation is critical for it’s function

As kidneys play a key role in RBC synth, kidney failure can lead to secondary anaemia

Renal tumours cause excess EPO secretion boosting erythropoeisis causing erythrocytosis

Too many RBC lead to infarcts (areas of tissue death) due to blood vessel blockages

EPO receptors are expressed in CFU-E & pronormoblasts.

Question 22

EPO molecular control

Answer 22

EPO gene has several segments that can sense levels of O (sensor regions)
Under control of a protein of a transcription factor called HIF - hypoxia inducing factor (HIF 2 alpha and beta) and others

Response to hypoxia (in rats) shows increase in EPO mRNA in kidney and liver. Within 60 mins of onset mRNA levels can be amplified >1000 fold in kidney (93% of production is in kidneys)

Question 23

Boosting performance/ cheating

Answer 23

Athletes can boost their performance by training at high altitudes where low O2 availability increases EPO production which goes to the bone marrow causing increase in RBC production

Cheating: Autologous, homologous transfusions, extract blood, freeze it then use it for transfusion before competition. However as blood is stored in plastic it is possible to test for residues (dioctyl phthalate) in blood

Other:
EPO injection (i.e. recombinant rhipo made in lab)
Prolyl hydroxylase inhibitor (PHD-I EG- 2216)
Erythropoeisis stimulation agents (ERAs) FG4592 (rodadustat)
Synthetic oxygen carriers

Question 24

Breakdown of blood

Answer 24

Blood is plasma and cells

Cells include
platelets (megakaryocyte fragments)
& RBC/ WBC

WBC can be split into
Granulocytes :
neutrophils, eosinophils, basophils
Agranulocytes:
lymphocytes - B cells and T Cells
monocytes - macrophages

Question 25

Neutrophils

Answer 25

Phagocytes and granulocytes
Make up 50-80% of circulating leukocytes
Have a single multilobed nucleus

Fine granules contain proteases & antimicrobial effector molecules such as defensins

Form the “front line of defence” the first layer of defense for an organism

Phagocytose bacteria in blood stream

Circulate for 7-10 hours in blood stream

Can leave blood stream by extravasation and infiltrate surrounding tissues remaining for a few days

Pliable nuclei, soft cells can squeeze through cell layers

12-15 micrometres in diameter

Migration to area of infection and ingestion of bacteria requires substances contained in granules

Primary granules contain: elastance & defensins & myeloperoxidase

Secondary granules contain: lysozyme, lactofferin, gelatinise and other proteases

Question 26

Eosinophils

Answer 26

Stain with eosin hence name

12-15 micrometre diameter

Phagocytic- mainly responsible for killing indigestible parasites by degranulating and dissolving the cell surface.

About 1-4% of circulating leukocytes are eosinophils

Large specific granules appear bright red and clearly discernible

Bilobed nucleus

Granular content:

Eosinophil peroxidase (EP) - binds to microorganisms and facilitates their killing by macrophage

Major basic protein (MBP) - main component of granules, binds to & disrupts membranes of parasites. Causes basophils to release histamine by Ca ²+ mechanism

Eosinophil cationic protein (ECP) - neutralises heparin, causes fragmentation of parasites

Eosinophil driven neurotoxin (EDN) - secretory protein with micronucleus+ antiviral activity

Question 27

Monocytes/macrophages

Answer 27

Make up 2-8% of leukocytes

Largest leukocyte 15-20 micrometres
Important in phagocytic defence

New monocytes circulate in blood for a few hours before migrating to tissues

In tissues become 5-10x larger and develop into active phagocytic cells - macrophages

Macrophage derived from monocytes are more efficient phagocytic cells than neutrophils

Question 28

Lymphocytes

Answer 28

Make up 30-40% of leukocytes

Range from 7-12 micrometres

Normally small, similar to RBC size
97% small and 3% large

Nucleus stains densely and is normally round or slightly indented

Nucleus occupies most of the cell reducing cytoplasm to a thin basophilic rim which occassionally has a few lysosomes.

B lymphocytes are produced in bone marrow

T lymphocytes are produced in bone marrow but mature in the thymus.
Less abundant class it T killer cells.

In fetal development yolk sac, liver and spleen are sites where lymphocytes originate from.

In postnatal life the bone marrow and thymus are the primary lymphoid organs where lymphocytes develop before exposure to antigens.
Secondary lymphoid organs are lymph nodes - the spleen and lymphoid aggregates of the gastro-intestinal & respiratory tracts.