Leukocyte development, kinetics, and function

- Leukocytes
 Colorless in appearance compared to RBCs in unstained specimen
 6 types in a microscope
 10 types in a flow cytometry
 Granulocytes – cytoplasm is filled with granules with different staining and are polymorphonuclear
 Agranulocytes (mononuclear cells) - does not have granules in their cytoplasm
- Lymphocyte is the true agranulocyte while monocyte contains granules
- Their nucleus is also round, folded, or indented
 Normal blood levels – 4.5 x10^9/L – 11x10^9/L
- Neutrophils
 They are created from the bone marrow
 HSCs (totipotent) -> pluripotent HSCs -> Myeloid progenitor (multipotent) -> GMP (granulocyte-
monocyte progenitor)
 HSC have a CD34 marker
 Share a common progenitor with monocytes GMP (kasama nya sa GMP lineage)
 G-CSF - Cytokine for stimulation of neutrophil production
 GCSF cytokine signals the GMP to become a neutrophil
 They are classified according to functioning pools:
 Stem cell pool – totipotent, pluripotent, multipotent that are capable of self-renewal and
differentiation
 These cannot be identified in microscopy but can be detected through flow cytometry
 Mitotic pool/Proliferation pool – it includes the CMP/CFU-GEMM (common myeloid
progenitor) -> GMP-> myeloblast, promyelocyte, myelocyte that are capable of mitosis
 Polycythemia vera increases all CFU-GEMM/CMP
 Rubricyte and myelocyte are the last stages capable of mitosis
 Maturation pool/Storage – they cannot mitose and are only contained for nuclear
maturation or storage ready to be released. These include metamyelocyte, band neutrophil
and segmented neutrophil
 As they mature, nucleus become smaller, nucleoli and rRNA disappear, chromatin
condenses, cytoplasm: nucleus ration becomes greater, granules appear
 Stages of neutrophil development
 Myeloblast – if there is positive nucleoli that is immature and the chromatin is fine
 0-3% of bone marrow cells
 14-20 micrometer in diameter
 Subdivided into 3 subclasses:
 Type 1 – high nucleus to cytoplasm ratio 8:1 or 4:1 w/ fine chromatin and 2-4
nucleoli with no presence of granules
 Type 2 – w/ presence of primary (azurophilic) granules that are fewer than 20 per
cell
 Type 3 – dark chromosome (condensed chromatin) and purple cytoplasm w/ more
granules
 Promyelocytes – presence of heterochromatin (condensened chromatin) and primary
granules seen as reddish in appearance
 BOARDS: first presence of primary granules
 1-5% of bone marrow cells
 16-25 micrometer in diameter
 Nucleus is round and eccentric
 Still contains 1-3 nuclewoli
 Presence of nuclear halo or “hof”
 Patients with Chronic promyelocytic leukemia has no nuclear halo
 Primary granules (MACPED)
  Myeloperoxidase – induces creation of oxide
 Acid b-glycerophosphatase – as phosphate source
 Cathepsins – proteases
 Proteinase-3 – causes neutrophilic inflammation
 Elastase – helps break down macromolecules as a digestive enzyme
 Defensins – killing or inhibiting bacteril growth

 Myelocyte –Final stage of neutrophil development capable of mitosis

 Final stage of neutrophil development capable of mitosis
 6-17% of bone marrow cells
 Synthesis of secondary granules and termination of creation of primary granules
 Divided into two subtypes:
 Early myelocyte – Dawn of neutrophilia - cytoplasm becomes pink and secondary
granules become evident in the golgi apparatus then cytoplasm becomes lavender-
pink due to spreading of lavender secondary granules then primary granules
decrease hence the reduction of reddish color
 Late myelocyte – becomes smaller (15-18 micrometer) and more heterochromatin
w/ lesser nuceloli
 Secondary granules: (BGCLTN)
 b2-Microglobulin – lymphoma biomarker
 Gelatinase – degrading gelatin
 Collagenase – degrading collagen fibers
 Lactoferrin – causes cell lysis when bound to LPS of bacteria
 Transcobalamin I (haptocorrin)– protects vitamin B12 from the stomach
 Neutrophil gelatinase-associated lipocalin (NGAL) – biomarker for infection

 Metamyelocyte- presence of slight indentation of the nucleus kindey shaped nucleus

 No longer capable of mitosis
 3-20% of bone marrow cells
 14-16 micrometer
 Synthesis of gelatinase/tertiary granules
 Absence of nucleoli and no basophilia
 Tertiary granules: (BGCLA)
 b2-Microglobulin – biomarker
 Gelatinase
 Collagenase
 Lysozyme – similar to lactoerrin
 Acetyltransferase – transfers acetyl CoA to lysine amino acids
 Neutrophilic stabs/bands –stab
 9-32% Most common cell in the bone marrow but 0-5%/ in the peripheral blood
 Indentation is half of the diameter if the indentation does not go beyond half of the
circle in meta myelocyte , it is metamyelocyte , if it goes past the line , it is a neutrophilic
band
 Synthesizes secretory granules
 Secretory granules:
Alkaline phosphatase – protease
 Segmented Neutrophils – there is segmentation of nucleus into 2-5 lobes
 7-30% of bone marrow cells
 50-70% in circulation or 2.3-8.1x10^9/L
 Continuous synthesis of secretory granules
 Lymphocytosis in neonates due to increasing development of neutrophils ( mas madami
lymphocytes in neonates than
  Synthesis : Primary -> secretory
 Secretion : Secretory -> primary ( last to be created, first to be released)

 Neutrophil kinetics – movement of neutrophils in the bone marrow precursors and in the circulation
 Production of 0.9-1.0x10^9/kg/day
 Multiplying pool – 2.1x10^9/kg/day
 Maturation – 5.6x10^9/kg/day
 Transit time : myeloblast to myelocyte : 6 days
 Maturation pool : 4-6 days
 Then G-CSF stimulates release of neutrophils to the circulation
 Subdivided into two pools in the circulation:
 Marginating neutrophil pool (MNP) – localized along the wall of the blood vessels for
ready immune function in case of infiltration (maraming MNP sa lungs)
 Usage of integrins and selectins for margination and diapedesis
 These are proteins that are located on the cell membrane that attaches to the ECM
of others cells while being attached to the internal cytoskeleton of the cell
 Circulating pool (CNP)
 Half-life - 7 hours in the blood example if you synthesize 1000 neutrophils, after 7 hours
500 will remain (gradual decrease)
 Neutrophil function
 Recognize non-antigenic matter
 Phagocytosis
 Release of chemotactic materials by microorganisms that initiates chemotaxis ->
adhesion by rolling of neutrophils on the endothelial cells with the use of selectin and
B2 integtins -> extravasation/diapedesis by integrins and collagenase/gelatinase – usage
of receptors to bind to foreign matter or opsonic materials -> engulfment using
pseudopodia and formation of phagosome -> inititation of respiratory burst creating
NADPH oxidase that creates ROS for antibacterial digestion like H2O2 converted to CLO-
by MPO -> Antigen presentation
 NET -Part of the complement system by releasing NET (neutrophil extracellular traps) and
allowing alternative pathway to happen
 NETs are formed from unfolded chromatin with primary and secondary granules that
can trap and kill microorganisms
 They can also release granules for chemotactic activity or for suicidal activity by
macrophages

- Eosinophil
 Characteristics
 Makes up 1-3% of the bone marrow and 1-3% in peripheral blood or 0.4X10^9/L
 Usually bi-lobed/ lung shaped
 Contain Basic granules (Eosin stain is acidic -> Basic granules)
 Eosinophil development
 Comes from CMP
 Mitotic pool consists of CMP, EBP, myeloblast, promyelocyte, myelocyte
 EBP is signaled by IL-3, IL-5, GM-CSF to become a eosinophil
 IL-5 is critical for eosinophil growth
 Promyelocyte – presence of Charcot Leyden crystals in primary granules
 Charcot Leyden crystals – Loeffler’s pneumonia ; presence of crystals due to
degranulation of eosinophil
 Myelocyte – presence of large reddish granules with azure granules in the cytoplasm
 Secondary granules are crystalline and has a crystal core
 Metamyelocyte and bands – presence of tertiary and secretory granules
 Secondary granules becomes more refractory/crystalline and increases in number
  It also creates Lipid bodies and small granules organelles
 Lipid bodies – contains all of the substrate needed for cyclooxygenase pathway
 Arachidonic acid (C20) - > COX enzyme 1 and 2 -> TXA2 ( for platelet activation)
 TXA2 is produced to signal platelet activation ; Aspirin inhibits COX enzyme to
prevent clotting of blood
 PGE2 is produced from COX enzyme to signal the hypothalamus to signal pain
( kaya paracetamol inhibits COX production to inhibit pain
 Mature eosinophils
 Bilobed and contains many refractory secondary granules
 Maturation time: from last myelocyte to the emergence of mature eosinophil is 3.5 days
 Eosinophil function
 Controls allergy due to having antihistamine
 Hallmarks of allergy - Can signify severity of asthma and allergic disorders
 Piecemeal degranulation- Phagocytosis then degranulation (kakagat tas dinuduraan ng
granule contents)
 They are antiparasitic (usage of Eosinophil cationic protein)
 Release Major Basic Protein (MBP) that causes Mast cell degranulation
 Can also be APC

Basophil
- Characteristics :
 True leukocytes that circulate and mature within the bone marrow
 Blood effector cell
 Contains water soluble granules
 Bi-lobed Contains Acidic granules (Methylene blue is basic -> acidic granules)
- Reasons why Basophils are few:
 Similar to neutrophils behaving when water is introduced , granules are removed kaya magkamuka
sila sa microscope (prone for dissolving of granule)
 If a granule is dissolved, it will leave a red rim on the vacuole
 Least amount approx.. 0-2% of circulation
 1% of bone marrow cells
- Basophil development:
 Also comes from MP
 Mitotic pool composes of: CMP , EBP
 EMP is signaled by GM-CSF and IL-3 to become a basophil
 Immature basophil – Already has a lobulated nuclei with condensed chromatin
 Presence of blue-black secondary granules
 Mature basophil – lobulated nucleus obscured by large irregular blue-black granules
 Colorless cytoplasm
 Lifespan is 60 hours
 Lifespan is the longer form most leukocytes because of anti-apoptotic measures activated by
IL-3
- Basophil function :
 Initiator/promoter of allergic Inflammation : Contain histamine (vasodilation and
bronchoconstriction as allergy) Leukotriene C4 (chemotactic) Heparan as heparin (natural
anticoagulant)
 Also induces B cells to release IgE
 Increase during allergy
 Can also release IL-4 and IL-13 that are Helper T cell cytokines that control their immune response
 Can release granzyme b and retinoic acid which are mediators of allergy and immune activity of NK
cells
 Angiogenesis – expression of vascular endothelial growth factor for creation of new blood vessels
Mast cell – same function with basophils but they do not have the same cell origin (Mast cell comes Mast cell
progenitors not GMP )
- Tissue effector cell
- They are not leukocytes
- MCP are released to the circulation then mature and become effector cells to the corresponding tissue
that they land on
 KIT ligand is required for growth of mast cells
- They are from the bone marrow but mature on tissues
- Function:
 Degranulation of granules leading to allergic inflammation
 APC
 Can also release cytokines for differentiation of T 2 helper cells
 They can also be anti-inflammatory and immunosuppressor

Monocytes
- Pseudo-agranulocyte because they still contain granules
- Monocyte development:
 Similar to Neutrophil development (CMP, GMP)
 GMP is signaled by MCSF to become a monocyte
 Mitotic pool – ( CMP, GMP, Monoblast, promonocyte, monocyte, macrophage)
 Promonocyte – First presence of indentation
 12-18 um in diameter
 Nucleus is slightly folded
 Fine chromatin
 1 nucleolus
 Smaller blue granules than promyelocytes composed of Peroxidase, esterase, and
lysozyme
 Monocyte – they tend to stick or to spread on glass or plastic
 15-20 um in diameter
 Their goal is to enter the tissue and become a part of the mononuclear phagocytotic
system composed of macrophages in different tissues
 Nucleus has deeper indentations like a horseshoe shape
 Looser chromatin that are stringy
 Absence of nucleoli
 Cytoplasm is blue with gray granules called Azure dust
- Monocyte kinetics
 60 hours for 1 promonocyte to undergo 2 mitotic divisions to produce 4 monocytes
 Increased demand can make it 16 monocytes in 60 hours
 Monocytes that are marginating pool are 3.5 times more than the circulating pool
 Monocytes remain in the circulation within 3 days
 Once in the tissue, they become macrophages

Macrophage – they become residents of their corresponding tissue

- Characteristics:
 Oval nucleus w/ reticular chromatin
 40-50um in diameter
 Cytoplasm is filled with debris or organism
 Lifespan depends on the tissue they reside in
 Mononuclear phagocytotic system :
 Liver- Kuppfer cells
 Lungs – alveolar macrophages
 Brain – microglial cells
 Skin – Langerhans cells
  Spleen – splenic macrophage
 Intestine – intestinal macrophage
 Bone – osteoclasts
 Peritoneum – peritoneal macrophage
 Kidneys- renal macrophages
 Lymph nodes – dendritic cells
- Functions :
 Responsible for chronic infection and fungal infection
 Innate immunity : Recognition of foreign matter by toll like receptors (TLRs) that stimulate
inflammation and phagocytosis.
 Can secrete nitric oxide that is cytotoxic
 Can phagocytose antibody or complement coated microorganism due to its fc and
complement receptors
 Adaptive immunity: Antigen presentation that initiates adaptive immune response
 Removal of cell debris and waste within the tissues

Lymphocytes
- Composed of T cells, B cells, and NK cells
- True agranulocyte because they do not contain granules
- Anamnestic response -They contain a specific surface molecule for a a specific antigen in order to
produce a memory immune response that is adaptive towards that antigen
 Second exposure of that antigen will cause a much faster, effective, and more aggressive immune
attack towards the foreign bodies containing the same antigen
- 3 characteristics of lymphocytes
1. They are not end cells , they are resting cells that are needed to be activated to become memory and
effector cells
 They are activated through antigenic exposure and presentation
2. They can recirculate outside and inside the circulation though the lymph and lymph nodes
3. Capable of rearranging their receptors genes to create a variety of antibodies and surface receptors
- Composes 18-42% of circulating blood cells / 0.8-4.8x10^9/L
- Lymphocyte development
 Non-antigenic development – occurs in the primary/central lymphatic organs which are bone
marrow and the thymus
RESTING CELLS
 Hematogones B Cells– immature b cells in lymphatic nodules that contain nuclear
chromatin and scantly cytoplasm
 9 um in diameter
 Chromatin is arranged in blocks
 T cells – develop in the thymic cortex as pro-T, pre-T, and immature T cells.
 They undergo gene variation to create T cell receptors that are unique to each T cell
 Then go to the thymic mediastinum to separate self-reactive T cell
 They comprise 51 – 88% of lymphocytes
 Antigenic development- occurs in the secondary/peripheral lymphatic organs which are lymph
nodes, tonsils, Peyer’s patch, appendix, and spleen
EFFECTOR CELLS
 Plasma cells – mature b cells in lymphatic nodules that came into contact with antigen
becomes an effector cell capable of creating antibodies
 Reactive lymphocytes/ variant lymphocytes – these are T cells that came into contact with
antigens
 Can be seen in patients that are experiencing an infection or had been cured recently of
such
- NK cells – large granular lymphocytes
 They are the only lymphocyte that has granules
  They contain CD 7, 13, 16, 57
 They contain azurophilic graules but do not have peroxidase
 Composes 4-29% of circulating lymphocytes
- Lymphocyte function
 TH1 – immune response against intracellular pathogens (viruses and bacteria)
 TH2 – immune response against extracellular pathogens (helminths and bacteria)
 Treg – regulates the bodies own immune response (siya nag cocontrol pag sobra na or kulang)
 CD8 cytotoxic T cells – secretes granzymes or perforins that causes cell lysis or activates apoptotic
pathway of a target foreign cell
 NK cell – part of innate immune system because it does not have an adaptive mechanism and does
not require pre-antigenic exposure
 It destroys tumor cells and virus infected cells through ADCC (antibody-dependent cell-
mediated-cytotoxicity) or also known as Fc receptor opsonization

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