Pages of 9-Leukemia 1 2.pdf

There are three main types of neutrophil granules: 1 2 4 3 Neutrophils have 3types of granules; primary (azurophilic) granules (found in young cells) and secondary (specific) granules...

There are three main types of neutrophil granules: 1 2 4 3 Neutrophils have 3types of granules; primary (azurophilic) granules (found in young cells) and secondary (specific) granules (which are found in more mature cells). The primary granules are surrounded by a phospholipid membrane and contain peroxidase and a number of antimicrobial compounds. myeloperoxidase (used to generate toxic bacteria-killing substances), is crucial for the production of hypochlorous acid, a powerful antimicrobial agent. Elastase: is a protease enzyme that breaks down elastin, a protein found in connective tissues. It contributes to the degradation of extracellular matrices, aiding neutrophils in tissue penetration and facilitating the immune response. Primary granules contain cationic proteins and defensens that are used to kill bacteria they disrupt the microbial cell membrane integrity, contributing to the elimination of pathogens. Cathepsin G to break down (bacterial) proteins. It has many functions. It can clear pathogens, regulate inflammation by modifying the chemokines, cytokines, cell surface receptors, control the blood pressure, and induce thrombogenesis. Lysozyme to break down bacterial cell walls. Bacterial Permeability Increasing Protein (BPI): it has both bactericidal and lipopolysaccharide-neutralizing activities. The present study suggests that BPI also plays an important role in phagocytosis of Escherichia coli by neutrophils through promotion of complement activation on the bacterial surface. Esterase N: It is an enzyme released by activated neutrophils after they have been recruited to sites of infection. Esterases hydrolyze the compounds that contain ester, amide, and thioester bonds, which cause prodrug activation or detoxification Secondary: (also known as specific granules)  Lactoferrin : An iron-binding protein that helps sequester iron , deprives microorganisms of iron and has anti-viral and antibacterial effects (a member of the transferrin family).  Lysozyme: is a hydrolytic glycosidase that can break down the cell walls of certain bacteria, contributing to the destruction of these pathogens, through its dual activities as a lytic enzyme and a small cationic protein  Histaminases: catalyze the breakdown of histamine important in controlling its effects and preventing prolonged or excessive responses specifically diamine oxidase (DAO) and histamine N-methyltransferase (HNMT), are two key enzymes involved in histamine metabolism  Collagenase: Facilitating the movement of neutrophils through tissues during the inflammatory response by degrading collagen. Gelatinase : Facilitating the movement of neutrophils through tissues to reach sites of infection or injury. They also contribute to the removal of damaged tissue during the resolution phase of inflammation refers to matrix metalloproteinases (MMPs), specifically Gelatinase A (MMP-2) and Gelatinase B (MMP-9). Gelatinase is a marker of terminal neutrophil differentiation-family of metalloenzymes known as matrix metalloproteinases (MMPs) or matrixins  Heparinase: is an enzyme that specifically degrades heparin by catalyzing cleavage of the saccharide bonds found in the heparin molecule, may modulate the anticoagulant effects of heparin Secretory vesicles are scattered throughout the cytoplasm of myelocytes, metamyelocytes, band neutrophils, and segmented neutrophils. are intracellular compartments that play a crucial role in regulating the interaction of neutrophils with endothelium during inflammatory responses. Secretory vesicles are formed by endocytosis in the later stages of neutrophil maturation and contain plasma proteins including albumin. When neutrophils are stimulated, the cytoplasmic secretory vesicles fuse with the plasma membrane to increase the neutrophil surface membrane and expression of adhesion and chemotactic receptors. Complement Receptor 1 (CR1):-Part of the upregulation of surface membrane proteins in response to inflammatory stimuli. Important for the interaction of neutrophils with endothelium. Cytochrome b558: Component of NADPH oxidase , Translocates to the plasma membrane upon vesicle mobilization.  Provides reduced oxygen metabolites for activation of gelatinase, crucial for the respiratory burst  Cytochemical Stains Myeloperoxidase (MPO) Sudan black B (SBB) Esterase Periodic Acid Schiff (PAS) Leukocyte Alkaline Phosphatase (LAP) Toluidine blue (for basophils- M8)  LAP-defending your body against viruses, bacteria, and other germs. They’re a crucial part of your immune system. In chronic myeloid leukemia (CML), you have less alkaline phosphatase Toluidine blue (also known as tolonium chloride) is an acidophilic metachromatic dye that selectively stains acidic tissue components (sulfates, carboxylates, and phosphate radicals). Toluidine blue (TB) has an affinity for nucleic acids, and therefore binds to nuclear material of tissues with a high DNA and RNA content Sudan Black- used for staining of neutral triglycerides and lipids. In addition to protein material found in neutrophilic granules, lipids and carbohydrates also can be found. About one-third of the lipids in neutrophils consists of phospholipids. Much of the phospholipid is present in the plasma membrane or membranes of the various granules. Cholesterol and triglycerides constitute most of the nonphospholipid neutrophil lipid. Cytoplasmic nonmembrane lipid bodies can also be found in neutrophils; their role in cell function is unclear. Lipid material is likewise found in neutrophilic precursors. A cytochemical stain for lipids, Sudan black B, is used to differentiate myeloid precursors from lymphoid precursors. In differentiating haematological disorders Sudan black will stain myeloblasts but not lymphoblasts DNA polymerase found in cell nucleus Positive in AML of erythroid leukemia (M6) Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells. Glycogen is also found in both neutrophils and some myeloid precursors. Neutrophils sometimes function in hypoxic conditions as at an abscess site and obtain energy by glycolysis, utilizing glycogen. The periodic acid-Schiff (PAS) stain is used to detect glycogen in cells. CD markers on the neutrophil include CD15, CD16, and CD11b/CD18.8 Neutrophil granulopoiesis and recruitment to the target site Granulopoiesis is characterized by the sequential formation of neutrophil granules. Myeloblast is the first cell of committed granulopoiesis that further differentiates into promyelocyte, myelocyte, metamyelocyte, band cell, and finally into mature neutrophil. Azurophilic or primary granules are synthesized at the promyelocytic stage. Specific or secondary granules are synthesized during the myelocyte stage and then gelatinase or tertiary granules are formed during the metamyelocyte stage. Finally, secretory vesicles (SVs), which are exocytoseable membrane-bound organelles, are formed at the late stage of neutrophil maturation. Mature neutrophils now egress from the bone marrow into circulation. Upon sensing any chemoattractant, mature neutrophils actively migrate from circulation to the site of infection or injury in a process called extravasation that is a multi-step process including rolling, adhesion, crawling, and transmigration  There are 3 populations of 1. Stem cell pool: within bone marrow - neutrophils: 90% A. B. 2. circulating pool within blood- 3% 3. Marginating pool: of neutrophils adherent to endothelium in low flow exchange vessels- 7% The mitotic pool, also called the proliferating pool, includes cells capable of DNA synthesis: myeloblasts, promyelocytes, and myelocytes. Cells spend about 3–6 days in this proliferating pool and undergo four to five cell divisions. The postmitotic pool, also known as the maturation and storage pool, includes metamyelocytes, bands, and segmented neutrophils. Cells spend about 5–7 days in this compartment before they are released to the peripheral blood. The number of cells in the postmitotic storage pool is almost three times that of the mitotic pool. Neutrophils are produced from stem cells in the bone marrow and spend about 1–2 weeks in this maturation compartment. Most neutrophils are released to the peripheral blood as segmented forms. When the demand for these cells is increased, more immature forms can be released. One-half of the neutrophils in the peripheral blood is in the marginating pool (MNP); the other half is in the circulating pool (CNP). Neutrophils spend hours in the blood before marginating and exiting randomly to tissue. Leukocyte extravasation, less commonly called diapedesis, is the movement of leukocytes out of the circulatory system and towards the site of tissue damage or infection. This process forms part of the innate immune response, involving the recruitment of non-specific leukocytes. Monocytes also use this process in the absence of infection or tissue damage during their development into macrophages. It has been estimated that they spend on average 4-5 days in the tissues before they are destroyed during defensive action or as the result of senescence. Leukocytes extravasation Adhesion of neutrophils to the vascular endothelium and eventual migration of neutrophils into the tissue occur as a result of activation of endothelial cells (EC) and neutrophils by exposure to chemoattractants. When the cells are activated, they are induced to express adhesion molecules. These transmembrane molecules send a signal across the membrane to the interior of the cell when they attach to their receptor. The process occurs in four stages: In Stage 1, E-, P-selectin and (VEC) receptor are expressed on activated EC. The neutrophil’s L-selectin and receptors for E-, P-selectin cause the neutrophil to attach loosely to the EC and roll along the endothelium. Neutrophils in Stage 2 are activated by the presence of chemoattractants in the local environment and express the b2 integrins. These chemoattractants also activate the Ecs (include specific and nonspecific proinflammatory mediators such as C5a (complement activation peptide), bacterial products, lipid mediators (e.g., platelet activating factor [PAF]), and chemokines). The neutrophils in Stage 3 attach to the activated ECs via the attachment of their b2@integrins to ICAMs of the EC, resulting in a firmer attachment than in Stage 1 and halting the rolling of the neutrophil. This induces a cytoskeletal and morphologic change in the leukocyte required for cellular migration. Stage 3 ends with a strong, sustained attachment of the leukocyte to the VEC. The neutrophil in Stage 4 migrates through the endothelium and basement membrane (diapedesis) to the area of inflammation. They move in the direction of the chemoattractants (chemotaxis). The neutrophils use pseudopods to squeeze between endothelial cells, leaving the vascular space and passing through the subendothelial basement membranes and periendothelial cells. Subendothelial basement membranes are presumably eroded by the secretion of the neutrophil enzymes gelatinase B and elastase from neutrophil granules. Migration is enhanced when IL-1 and/or TNF activate the VEC.1 Stage 1 involves the activation of VECs that allows for a loose association of VECs with neutrophils. Inflammatory cytokines induce VECs to express of E- and P-selectins and L-selectin ligand. E- and P- selectin molecules on the VEC surface interact with their ligands on the neutrophil. Additionally, L-selectin, which is constitutively present in the neutrophil membrane, interacts with its L-selectin ligands that are upregulated on the surface of the activated VEC. These interactions induce the neutrophil to transiently associate and dissociate with the VEC, causing the neutrophil to “roll” on the VEC surface. The rolling neutrophils are thus situated to respond to additional signals from chemoattractants (chemotactic substances—chemical messengers that cause directional migration of cells along a concentration gradient) generated by infectious agents or an inflammatory response. Stage 2 is the activation of neutrophils. Chemokines (cytokines with chemotactic activity) or other chemoattractants bind to the endothelial cell surface where they interact with the loosely bound neutrophils and result in activation of neutrophil integrins. Chemoattractants are released by tissue cells, microorganisms, and activated VEC and include specific and nonspecific proinflammatory mediators such as C5a (complement activation peptide), bacterial products, lipid mediators (e.g., plateletactivating factor [PAF]), and chemokines. Upon neutrophil activation, activation-dependent adhesion receptors including the b2@integrin molecules are expressed. Leukocyte plasma membranes have at least three b2@integrins: CD11a/CD18 (leukocyte function associated antigen-1 [LFA-1]), CD11b/CD18 (Mac-1, also known as the complement receptor 3 [CR3]), and CD11c/CD18. Each has an a subunit (CD11a, CD11b, CD11c) noncovalently linked to a b subunit (CD18).10 The neutrophil molecule, L-selectin, is downregulated at this time. Stage 3 involves arrest of neutrophil rolling because the activated neutrophils are more tightly bound to the VECs. Expression of activation-dependent b2@integrin adhesion molecules on neutrophils mediates firm adherence to ICAMs expressed by activated VECs near the site of infection or inflammation. This induces a cytoskeletal and morphologic change in the leukocyte required for cellular migration. Stage 3 ends with a strong, sustained attachment of the leukocyte to the VEC. At this time, leukocyte NADPH oxidase membrane complexes are assembled and activated (discussed in the section, “Bacterial Killing and/ or Digestion”). Stage 4 involves the transendothelial migration phase that occurs when neutrophils move through the vessel wall at the borders of VECs by the process of diapedesis. As neutrophils pass out of the vessel and into the tissue, VECs modify their cell-to-cell adherent junctions. The neutrophils use pseudopods to squeeze between endothelial cells, leaving the vascular space and passing through the subendothelial basement membranes and periendothelial cells. Subendothelial basement membranes are presumably eroded by the secretion of the neutrophil enzymes gelatinase B and elastase from neutrophil granules. Migration is enhanced when IL-1 and/or TNF activate the VEC.10