HC23: Normal hematopoiesis

Blood cells and functions

Blood composition:

Blood forms 8% of the total body weight, the other 92% consists of fluids and tissues. Blood is made up for 55% of blood plasma and for 45% of formed elements. Plasma is mostly made up of water, formed elements of red blood cells, white blood cells and platelets.

Main functions:

Blood has many functions:

• Homeostasis: regulation of body temperature by plasma
• Body supply: oxygen and nutrients supply by erythrocytes and plasma
• Waste collection: of carbon dioxide and lactic acid by erythrocytes and plasma
• Defense: anti-microbial and anti-tumor response by leukocytes
• Coagulation: by thrombocytes

Erythrocytes:

Erythrocytes form the largest fraction of blood cells → there are 5 x 10¹²erythrocytes per liter blood. They are red cells → cause the red color of blood. Erythrocytes can spend up to 120 days in the circulation. They have no nucleus and are fully differentiated → no proliferation takes place. Their function is oxygen transport.

Erythrocytes contain hemoglobin:

• Males: 8,5-11,0 mmol/L
• Females: 7,5-10,0 mmol/L

Leukocytes:

The normal leukocyte count in blood is 4-10 x 10⁹ per liter. Leukocytes can differentiate into:

• Granulocytes: have a life span of 1-2 days
  • Neutrophilic granulocytes
    • 1,5-7,5 x 10⁹/L
    • Defense against encapsulated bacteria
  • Eosinophilic granulocytes
    • <0,5 x 10⁹/L
    • Mediate allergic responses and defense against parasites
  • Basophilic granulocytes
    • <0,2 x 10⁹/L
    • Mediate allergic responses and defense against parasites
• Lymphocytes: have a life span of days-years
  • B-cells
    • Antigen-specific immune defense
      • Antibody production
      • Cytokine secretion
  • T-cells: CD4 T-cells and CD8 T-cells
    • Antigen-specific immune defense
      • Cellular cytotoxicity
      • Cytokine secretion
  • NK-cells
    • Antigen-independent defense
      • Tumor surveillance
    • Cytotoxic capacity
• Monocytes: have a life span of 12 hours
  • Phagocytosis
    • Bacteria
    • Cell debris
  • Secretion of cytokines
    • TNF
    • IL-1
  • Antigen processing and presentation to T-lymphocytes
  • Macrophages are monocytes that reside in the tissue

Thrombocytes:

Thrombocytes are present in the blood in a concentration of 150-400 x 10⁹/L. They cause coagulation in case of vessel damage:

1. Injury to vessel lining triggers the release of clotting factors called von Willebrand factors
2. Thrombocytes bind to the von Willebrand Factors
3. The coagulation cascade is activated
4. Fibrin strands adhere to the plug to form an insoluble clot
5. Blood clots are made and the vessel wall is repaired again

Thrombocytes have a lifespan of 8-10 days.

Dally production:

Elements are in constant turnover and have different lifespans:

• Erythrocytes: 150 x 10⁶ per minute
• Granulocytes: 50 x 10⁶ per minute
• Thrombocytes: 150 x 10⁶ per minute 

Production must react to rapid changes in the environment to ensure homeostasis. Only mature elements gain access to the circulation. In case of infection or bleeding, production can increase 3-8 folds.

Stem cells

Stem cells are capable of self-renewal, the ability to go through numerous cycles of cell division while maintaining an undifferentiated state. They proliferate and differentiate:

• Proliferation: capacity to divide and proliferate
• Differentiation: the capacity to differentiate into specialized cell types

Types of stem cells:

There are 3 types of stem cells:

• Omni-potential stem cells: can differentiate into any type of cell
  • An embryonic cell
• Pluri-potential stem cells: can differentiate into more than 1 type of cell
  • E.g. a cell which can only become a type of blood cell
• Committed stem cells: can only differentiate into 1 type of cell
  • E.g. a cell which can only become an erythrocyte

During differentiation, cells lose their capacity to proliferate → fully differentiated cells will not proliferate and eventually die.

Hematopoietic stem cells:

Hematopoietic stem cells have the capacity for self-renewal. They can differentiate into all of the lympho-myeloid lineages → they are pluri-potent stem cells. Hematopoietic stem cells have symmetrical and asymmetrical division:

• Symmetrical division: cells become the same thing
• Asymmetrical division: cells become other things
  • 1 cell differentiates
    • Cannot go back to its previous state again
  • 1 cell is used for self-renewal

Hematopoietic stem cells are not microscopically identifiable in the bone marrow. Their estimated presence is about 2-5:10.000-15.000.

At the top of the stream of the hematopoietic system, more self-renewal is present, while further down there is more proliferation and differentiation.

Hematopoiesis

Embryonic development:

Hematopoiesis starts around the third week of pregnancy:

1. 3-11 weeks: islands of hematopoiesis are present in the yolk sac
2. 6-24 weeks: hematopoiesis takes place in the liver, and for 20% in the spleen
3. After 11 weeks: gradual hematopoiesis in the bone marrow
4. After 24 weeks: the bone marrow is the dominant source of hematopoiesis

After birth, hematopoiesis mostly takes place in the vertebra and pelvis.

Erythropoiesis:

Erythropoiesis starts as follows:

1. Stem cell: a hemocytoblast
2. Committed cell: proerythroblast
3. Developmental pathway
   1. Phase 1: ribosome synthesis → early erythroblast
   2. Phase 2: hemoglobin accumulation → late erythroblast → normoblast
   3. Phase 3: ejection of nucleus → reticulocyte
   4. Erythrocyte

This doesn’t have to be known completely by heart. The process mainly takes place in the bone marrow compartment. Only the end of phase 3 and the creation of the erythrocyte itself takes place in the blood. Erythrocytes have no nucleus.

Erythrocytes transport oxygen:

1. Oxygen arrives in the lungs
2. Oxygen binds to hemoglobin molecules in the erythrocytes
   • Each erythrocyte contains several 100.000 hemoglobin molecules which transport oxygen
   • Oxygen binds to heme on the hemoglobin molecule
   • Hb + O2⟷HbO2
3. Oxygen is released to tissue cells

Granulopoiesis:

Neutrophilic granulocytes create immunity against bacteria by inducing phagocytosis and releasing cytotoxic factors from granules. They form 40-75% of leukocytes, which increases to 10-30x as much during infection. Neutrophils circulate in the blood for 6-10 hours, and eventually migrate to tissues.

Neutrophilic granulocytes develop as follows:

1. Myeloblast
2. Promyelocyte
3. Myelocyte
4. Metamyelocyte
5. Band neutrophil
6. Segmented neutrophil

These phases do not have to be known by heart. During infection, a left shift becomes present. A left shift is the presence of more immature forms of neutrophils in the blood due to increased production.

Lymphopoiesis:

During lymphopoiesis, B- and T-cells are produced:

• B-cells
  • Recognize circulating proteins (antigens)
  • B-cells develop in the bone marrow
    • Hematopoietic stem cell → immature B-cell → mature naïve B-cell → activated B-cell → germinal center B-cell → long lived plasma cell of memory B-cell
      • Affinity maturation takes place in germinal centers of lymphoid organs
      • Memory B-cells can live for year in the body
      • Plasma cells produce antibodies
• T-cells
  • Don’t recognize circulating proteins, but recognize peptides presented by MHC-molecules from antigen presenting cells
  • T-helper cells and cytotoxic T-cells have an important role in cellular immunity
  • T-cells partly develop in the bone marrow and are educated in the thymus

1. Positive selection: T-cells bind to self MHC
   • Selection based on what the T-cells can recognize
2. Negative selection: elimination of T-cells recognizing self-antigens
3. Result: T-cells are capable of recognizing foreign peptides in self-MHC

Both fully mature T-and B-cells travel to the lymph nodes, where they meet each other. Here, T-helper cells (also known as CD4 T-cells) help activate B-cells:

1. An antigen presenting cell presents an antigen with its MHC-II molecule
2. T-helper cells get activated → produce cytokines which activate B-cells
3. B-cells differentiate into plasma cells and memory B-cells

Cytotoxic T-cells are also known as CD8 T-cells and have a direct effect:

1. An antigen presenting cell presents an antigen with its MHC-I molecule
2. An immature T-cell goes into clonal selection → forms activated cytotoxic T-cells and memory T-cells
3. Activated cytotoxic T-cells produce toxic granules which target the cell

NK-cells are capable of killing foreign cells. They have no antigen specific receptor, but can kill the cell in case of missing self or induced self:

• Missing self: no MHC or other self-presenting peptides are present
• Induced self: something is wrong with the cell or too many cell-surface molecules are present

Thrombopoiesis:

Thrombocytes are formed by the fragmentation of megakaryocytes. Megakaryocytes are giant cells with multiple copies of DNA in the nucleus. The edges of megakaryocytes break off to form cell fragments called platelets. Thrombocytes have a life span of 7-10 days.

Growth factors and cytokines

The difference between growth factors and cytokines is important:

• Growth factors can directly influence hematopoiesis via inhibition or stimulation of hematopoietic stem or progenitor cells
• Cytokines can indirectly influence hematopoiesis via inhibition or stimulation of production of hematopoietic growth factors

A few growth factors that affect multipotential cells are:

• EPO
• GM-SCF: gran/mono colony stimulating factor
• Thrombopoietin

Erythropoietin:

Erythropoietin (EPO) reduces the level of cell-cycle inhibitors. EPO augments the transcription of cyclins and supports their survival by increasing the anti-apoptosis protein BCLXL. EPO is mainly produced in the kidney.

EPO can be applied subcutaneously to patients with renal anemia or myelodysplasia.

Granulocyte-colony stimulating factor:

Granulocyte-colony stimulating factors (G-CSF) are produced in:

• Endothelial cells
• Fibroblasts
• Macrophages

Activated monocytes secrete inflammatory cytokines:

• Tumor necrosis factor α (TNF-α)
• Interleukin-1
• Interleukin-6

These cytokines stimulate G-CSF production → leukocytosis in patients suffering from infection or inflammation.

G-CSF can be applied subcutaneously to patients suffering from granulocytopenia, for instance after chemotherapy.

Thrombopoietin:

Thrombopoietin (TPO) is a stimulator of platelet production. It mainly is produced in the liver.

TPO also supports the survival and proliferation of hematopoietic stem cells, leading to enhanced expression or nuclear localization of several transcription factors:

• STAT
• Homeobox B4
• Homeobox A9

TPO can be given subcutaneously or orally to patients with ITP.

Stem cell mobilization

G-CSF induces increased neutrophil production and activation, which leads to release of cytokines such as elastase and cathepsin-G to facilitate mobilization. The binding between hematopoietic stem cells and the micro-environment is cut, making it possible for the cells to leave the bone marrow and circulate in the blood. This way, donor tissue can be extracted without actually invading the bone marrow.