WBC Development, Kinetics & Functions PDF

Leukocyte Development, Kinetics & Functions White blood cells (WBCs) Colorless Types Staining Surface antigens -Romanowsky stain -Flow cytometry -6 types -10 types  Granulocytes Cytoplasm are filled with granules...

Leukocyte Development, Kinetics & Functions White blood cells (WBCs) Colorless Types Staining Surface antigens -Romanowsky stain -Flow cytometry -6 types -10 types  Granulocytes Cytoplasm are filled with granules Mononuclear cells Differs in staining characteristics Nuclei Nuclei Do not segment Segmented or lobulated Round Neutrophils Oval Granules reacts with both acid & basic stains Indented Pink - lavender Folded Polymorphonuclear cell/PMN Monocytes & Lymphocytes Eosinophils Granules contain basic protein with acid stains (eosin) Orange red - red Basophils Granules contain acidic protein with basic stains (methylene blue) Blue - blue black  Functions: Number of circulating leukocytes varies Mediates immunity Sex Innate (nonspecific) Age Phagocytosis Activity Neutrophils Time of day Adaptive (specific) Ethnicity Production of Ab stress Lymphocytes being consumed Plasma cells being destroyed Kinetic: BM production the movement of cells through: Reference interval Development stages 4.5 – 11.5 X 109/L Into circulation From circulation to tissues Time in each phase  NORMAL MATURATION CHARACTERISTICS OF GRANULOCYTES MYELOBLAST Earliest morphogically identifiable granulocytic precursor 0 – 3% of the nucleated cells in BM 14 -20 um in diameter Type I High N:C ratio = 8:1 – 4:1 Slightly basophilic Fine chromatin Type III 2-4 nucleoli Darker chromatin No visible granules More purple cytoplasm Type II Contains more than 20 granules Dispersed azurophilic granules Do not obscure the nucleus Not exceeding 20 Rare in normal BM AML PROMYELOCYTE 1 – 5% of the nucleated cells in BM 16 -25 um in diameter Nucleus Cytoplasm Round to oval; eccentric Evenly basophilic hof prominent azurophilic granulation Paranuclear halo enzymes seen in normal cells myeloperoxidase more condensed chromatin pattern chloroacetate esterase 1-3 nucleoli MYELOCYTE recognizable appearance of secondary or specific cytoplasmic granules 6 – 17% of the nucleated cells in BM neutrophilic granules Final stage of cell division fine 15 – 18 um in diameter blue-pink Nucleus eosinophilic granules more oval larger nucleoli is no longer visible orange more clumped chromatin glassy or semi-opaque texture pattern (heterochromatin) basophils dark blue-black color dense METAMYELOCYTE Cytoplasm 3–20% of the nucleated cells in BM Start of the synthesis of Gelatinase 14 – 16 um in diameter granules Cell is incapable of division Residual RNA Nucleus Little or no basophilia indented or kidney bean-shaped secondary or specific cytoplasmic more clumped chromatin pattern granules is the major distinguishing Nucleoli is absent feature Granular leukocytes NEUTROPHIL Segmented & Band 3. Maturation pool Developmental Pools (BM) Storage pool 1. Stem cell pool Marrow reserve HSCs Consisting of cells undergoing Capable of self-renewal and nuclear maturation differentiation Metamyelocyte 2. Proliferation (Mitotic) pool Band neutrophil Consists of cells that are dividing Segmented neutrophil CMP/CFU-GEMM GMPs Major Cytokine Myeloblast G - CSF Promyelocyte Myelocyte  Mature form Band neutrophils 9 –32% of the nucleated cells in BM Segmented neutrophils 0-5% of the nucleated peripheral cells 7 –30% of the nucleated cells in BM Evidence of RNA is absent multilobed nucleus Synthesis of tertiary granules lobes are attached by a fine thread- Start of synthesis of secretory granules like filaments Nucleus: 2 – 5 nuclear lobes Highly clump 50 -70% of the nucleated peripheral cells Indented 2.3 – 8.0 x 109/L Exceeds ½ of the diameter of the nucleus The nucleus must be the same diameter throughout its length “when doubt exists, the cell should be called a segmented neutrophil”  Movement between the BM, peripheral blood & tissues Production Neutrophil Kinetics 0.9 – 1.0 x 10 cells/kg/day 9 Proliferative pool 2.1 x 109 cells /kg Myeloblast 6 days myelocyte Factors that regulates apoptosis: Maturation pool Pro & Anti-apoptotic 5.6 x 109 cells/kg (5 day supply) Bcl-2 family Transit time = 4 – 6 days Anti-apoptotic G-CSF (Stimulates the release of granulocytes from BM Mcl-1 Division of peripheral blood neutrophil Myeloperoxidase Circulatory neutrophil pool (CNP) Both are products of Marginated neutrophil pool (MNP) inflammation & infection Localized in walls of capillaries in tissues (Spleen, liver, lungs) Prolong neutrophil lifespan ❖ Integrins & selectins Apoptotic signal allows neutrophils to exit blood and enter tissues diapedesis MAC-1 Half – life = 7 hours (blood) Triggers death & phagocytosis  Neutrophil Function Part of innate immune system Characteristics of innate immune system Destruction of foreign organism that is not Ag specific No protection against reexposure Major Functions Reliance to barriers 1. Phagocytosis Inclusion of humoral system (complement system) Chemotaxis Motility Diapedesis 2. Destruction of foreign materials & microorganism Engulfment Digestion 3. Generation of NETs(neutrophil extracellular traps) 4. Secretory function Process of Phagocytosis ✓ Phagocytes engulf and kill microorganisms ✓ Steps of phagocytosis: 1. Chemotaxis 2. Adherence: Recognition and attachment 3. Ingestion: Engulfment and creation of phagosome 4. Digestion: o Fusion of phagosome w/ lysosome o Destruction and digestion o Residual body – Exocytosis  Chemotaxis cells are guided to the site of injury by the Rolling of neutrophils: concentration gradient of chemotactic substances Adhesive molecules on EC surfaces: Extravassation (pseudopodia) P/E-Selectins Rolling, adhesion, crawling, transmigration appeared under the influence of Diapedesis inflammatory mediator Amoeboid movement of migration of neutrophil Β2 Integrin through the vessel wall to the Interstitial tissues Leukocyte-specific CD11b/CD 18 integrin Recruitment begin when chemotactic agent binds to Contributes tight stationery binding neutrophil receptors. between neutrophils & EC Chemotactic agent Binding of ligand result to: Microorganisms Activation of outside–in signaling pathways Damaged cells Stabilizes adhesion Other leukocytes Initiates cell motility Responses Active crawling Roll along endothelial cells ICAM-1 (EC) MAC-1(neutrophils) Secrete granules containing adhesive molecules are fused to neutrophil plasma Neutrophil migrates in a directional manner membrane towards the greatest conc. of chemotactic agent Diapedesis Digestion ❑ Formation of phagosome allows the assembly of NADH oxidase complex (NOX2) Generation of reactive oxygen species (H2O2) H2O2 MPO Hypochlorite Respiratory Burst oxygen-dependent mechanisms ❑ Metabolic changes = fusion of primary & secondary granules to the phagosome Release of bactericidal molecule ❑ Killing or degradation Oxygen-dependent mechanisms o Fusion of primary & secondary granules fuse to phagosome o Release of bacterial molecules into phagosome o Tertiary granule degrade the extracellular matrix o Gelatinase ✓ degrades denature collagen (IV & V) ✓ Activates chemokines (IL-8) o Chemotactic agent ✓ Extravassation & migration of more neutrophils Generation of NETs Neutrophil extracellular traps Extracelluar threadlike structure enzymes Trap & kill gm(+) & gm (-) bacteria Fungi Generated at the time of death caused by antibacterial activity NETosis - Describes the unique form of neutrophil cell death that results to the release of NETs Secretory function Transcobalamin 1/R binder protein Necessary for Vit. B12 absorption Cytokines EOSINOPHIL EOSINOPHILIA 1% - 3% of the nucleated cells in BM Infection by parasitic helminths ¼ eosinophilic metamyelocyte Allergic disorder ¾ eosinophilic promyelocyte or Eosinophilia - hallmark eosinophilic myelocyte Asthma 1% - 3% peripheral blood # of eosinophil in blood & Absolute count = 0.4 x 109/L sputum correlates with disease severity EOSINOPHIL DEGRANULATION (Function) Airway remodelling Exocytosis Increase in thickness of airway wall Granules fuse with plasma membrane & empty content Eosinophil-derived fibrogenic to extracellular space growth factor Compound exocytosis Gastrointestinal tract Granules fuse together within eosinophil Food allergy Piecemeal degranulation Allergic colitis Secretory vesicles remove specific proteins from Crohn’s disease secondary granules vesicles migrate to the plasma membrane & fuse to empty the specific proteins to the extracellular space EOSINOPHIL DEVELOPMENT IL -3 ❑ Eosinophilic metamyelocyte IL-5 Secondary granules Most critical for growth & survival Distinct & refractory GM-CSF Secretory granules/vesicles (3rd type) Transcription factors GATA-1 PU.1 ❑ Mature Eosinophil c/EBP Bilobed nucleus ❑ Eosinophilic promyelocyte Refractile red-orange secondary granules Charcot – Leyden crystal protein The 1st identifiable eosinophilic precursor is Cytochemical early myelocyte ❑ Eosinophilic myelocyte Large, pale reddish-orange secondary granules Blue cytoplasm TEM Granules contain electro-dense crystalline core Eosinophil Kinetics IMMUNE REGULATION Deletion of double-positive thymocytes Myelocyte mature eosinophil (thymus) 3.5 days Eosinophil migrates to the thymus of Mean turnover 2.2 x 109 cells /kg/day newborns Storage pool = 9 - 14 x 109 cells /kg Acts as antigen presenting cells Half-life = 18 hours (circulation) Promote proliferation of effector T-cells Tissues = 2 – 5 days Initiate either type 1 or 2 immune responses Underlying columnar surfaces Rapidly secrete preformed cytokines Respiratory REGULATE MAST CELL FUNCTION Gastrointestinal Release MBP to cause mast cell Genitourinary Tracts degranulation Cytokines production Nerve growth factor production Promotes mast cell survival & activation BASOPHIL < 1% of the nucleated cells in BM ❑ Immature basophil 0% - 2% peripheral blood Round to lobulated nuclei DEVELOPMENT Slightly condensed chromatin IL-3 Blue cytoplasm with large blue-black Lifespan = 60 hrs granules INNATE & ADAPTIVE IMMUNITY Water soluble Cytokines for subtype 2 helper T-cells ❑ Mature basophil IL-4 lobulated nuclei IL-13 Clumped chromatin Regulate Th2 immune response Colorless cytoplasm with large blue-black Induce B cell to synthesize IgE granules initiator of allergic inflammation Dissolved granules leave a reddish-purple Angiogenesis rim Vascular endothelial growth factor (VEGF) Controls helminth infection  DEVELOPMENT MAST CELL Mast Cell Progenitors (MCPs) BM tissue effector cells Spleen Cytokine for maturation & differentiation allergic responses Kit Ligand (stem cell factor) Inflammatory reaction 1.The precursor circulate in FUNCTIONS the peripheral blood on Effector cell in allergic reaction the way to tissue destination Antigen-presenting cell 2.Phenotypic & functional induce the differentiation of Th2 cells similarities with Mediator of innate & adaptive immunity basophils & eosinophils Anti-inflammatory & immunosuppressive functions Immunologic “gatekeepers” Location Role in barrier function