Microbiology 435 Granulocytes

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Neutrophils Differentiation, proliferation and maturation takes place in the red bone marrow. Developmental stages: myeloblast promyelocyte myelocyte metamyelocyte band segmented neutrophil  Proliferative stages include the first three stages. The myeloblasts, promyelocytes and myelocytes undergo mitosis as they mature. By the time a cell has reached the myelocyte stage it has undergone 4 or 5 cell divisions. Maturation & storage stages includes the last three stages. General maturational changes that can be seen are: Overall cell size generally decreases as the cell matures. The nucleus decreases in size and starts to indent at the metamyelocyte stage and is segmented into up to 5 lobes at the segmented neutrophil stage. The nuclear chromatin pattern changes fine to coarse as the cell matures. The coarseness indicates mitotic inactivity (no more mitosis). Nucleoli disappear by the myelocyte stage. Nucleoli are composed of RNA and are associated with cell division. The amount of cytoplasm increases as a cell matures. (N:C ratio decreases from 4:1 to 1:1) Primary (nonspecific) granules start to appear at the promyelocyte stage. Secondary (specific) granules start to appear at the myelocyte stage. Overall cytoplasmic color goes from blue to neutral. The intensity of blue of the cytoplasm indicates the amount of RNA present. Distribution of neutrophils in adult peripheral blood ranges from 60-80% neutrophils (6.8 x 109/L). The life span of a neutrophil can be as long as a few weeks. Maturational changes at each stage 1. Myeloblast large cell large round nucleus fine chromatin pattern nucleoli blue cytoplasm no granules 2. Promyelocyte larger than myeloblast (exception to maturation rule) large round nucleus chromatin pattern getting coarser chromatin may be hard to see because of granule production non-specific granules, primary granules (red) are produced 3. Myelocyte cell getting smaller nucleus round to flattened, coarser chromatin N:C ratio getting smaller (more cytoplasm) cytoplasm is less intensely blue specific secondary granules begin appear 4. Metamyelocyte nucleus indents up to 50% of nuclear diameter chromatin becomes coarser secondary granules are predominant may be a few residual primary granules 5. Band nucleus is greater than 50% indented eventually will become about the same width all along its length chromatin is coarse Neutrophils can be divided by their nuclear structure into bands and segmented cells. There are two rules which can be applied to discriminate the two forms. Rule of Thread: As soon as the nucleus has a thread like indentation it is called segmented Rule of Third: As soon as the nucleus has an indentation of more then one third of the largest diameter it is called segmented. 6. Segmented neutrophil nucleus generally has 3 to 5 segments chromatin is very clumped Neutrophils can be divided by their nuclear structure into bands and segmented cells. There are two rules which can be applied to discriminate the two forms. Rule of Thread: As soon as the nucleus has a thread like indentation it is called segmented Rule of Third: As soon as the nucleus has an indentation of more then one third of the largest diameter it is called segmented. What’s happening as the cells change in appearance? The nucleus in immature cells is large because it is physiologically active. The nucleus contains euchromatin (light, loose, active with nucleoli) if the cell is engaged in mitosis. The nucleus contains heterochromatin (dark, clumped, inactive) if the cell cannot divide. Cytoplasm will be rich with RNA (blue) if a lot of protein synthesis is occurring (The RNA produces protein such as antibodies, cytokines or proteins associated with cell division). If there is an abundance of protein production, the Golgi Apparatus (part of the endoplasmic reticulum that prepares the protein for secretion) will be very prominent as a clear area near the nucleus (perinuclear clear area). The specific cytoplasmic granules will have various shapes and colors dependent on their content. Eosinophils and Basophils Eosinophils normally comprise about 3% of circulating WBCs. Basophils normally comprise about 0.5% of the circulating WBCs The maturation sequence is similar to neutrophils but only 2 or 3 segments form at the mature stage. Secondary granules are different color; eosinophils have red granules, basophils have deep purple to black that may obscure the nucleus and/or cytoplasm. Basophil granules are water soluble and may wash out in poorly fixed smears and appear as empty areas in the cytoplasm. This gives the cytoplasm a white appearance.   Functions of Granulocytes Neutrophils Neutrophils respond to an inflammatory reaction caused by biological, chemical or physical assault such as heat, cold, excess sunlight, stress, physical trauma. Neutrophils are attracted to the site of inflammation by chemotaxis, chemical signals sent out by injured cells or by microbes. They are highly motile and leave through the capillary walls by diapedesis and migrate to the tissue site. If the inflammatory reaction is severe enough, initially there may be a neutropenia. This can be followed by a neutrophilia, and a resulting left shift. Functional development of neutrophils 1. Myeloblast-essentially a nonfunctional cell. 2. Promyelocyte - primary granules contain myeloperoxidase, an enzyme required for intracellular microbial killing, and other enzymes. 3. Myelocyte - cell acquires some motility. The secondary granules that begin to be produced during this stage contain important compounds. Alkaline phosphatase is one of these compounds. It can be detected using a special stain used to differentiate CML from leukemoid reactions. 4. Metamyelocytes - cytoplasm contains both primary and secondary granules which contain compounds necessary to kill and degrade foreign agents (toxic, infectious or non-self agents); A metamyelocyte still can’t respond to chemotactic factors and can’t initiate phagocytosis. 5. Band - possesses full motility, active adhesion properties (needed for phagocytosis) and some phagocytic abilities. They are almost mature neutrophils. 6. Segmented (mature) - the fully functional neutrophil, actively motile and able to respond to chemotaxis. The primary function of neutrophils is phagocytosis. Phagocytosis routinely takes place in the respiratory system, the gastrointestinal system and the urinary system. It is the process by which cells engulf and disable particles. Toxic change: appearance of neutrophils when they are active against severe bacterial infections includes toxic granulation, vacuolization of the cytoplasm, Dohle bodies, and left shift. This is a typical reaction characterized by an increase in digestive enzymes and digestive vacuoles with an increased RNA activity. Steps of phagocytosis: Opsonization (Greek = to prepare for dining) Neutrophils cannot efficiently recognize and attach to most microbes; this process marks the organism for ingestion by coating the particle with immunoglobulin (antibody) and complement (a series of proteins that cause disruption of the bacterial membrane). Neutrophil membrane pseudopods envelop the microbe forming a vacuole called a phagosome within the cytoplasm. The phagosome fuses with lysosomal granules from the neutrophil’s cytoplasm. The granules release lytic enzymes into the phagosome. The lytic enzymes lead to eventual killing and digestion of the foreign agent. All these processes require energy that is derived by anaerobic glycolysis (glucose breakdown). One of the products formed in the digestion of the foreign particle is hydrogen peroxide, which is capable of killing microorganisms. Myeloperoxidase, one of the enzymes in the primary granules, catalyzes a reaction involving H2O2 resulting in a more toxic product. Myeloperoxidase deficiency is reported to be the most common congenital neutrophil disorders. However, the condition is fairly benign except for patients with other problems, like diabetes where fungal infections may occur. Function of basophils Basophils play a role in acute allergic reactions. Their granules contain histamine, heparin and other substances that are released in response to the presence of allergens. These substances cause increased vascular permeability, smooth muscle spasm, vasodilation, and the clinical symptoms of an allergic reaction: watery eyes, runny nose, and difficult breathing. Histamine is a vasodilator that makes blood vessels more permeable. This effect is usually seen at inflammatory sites and allows increased cellular movement through the vessel walls. Heparin prevents blood clotting. Both histamine and heparin enhance the migration of leukocytes to the inflamed site. Function of eosinophils Eosinophil granules contain enzymes, cytotoxic proteins and cytokine mediators. Eosinophils are found in high numbers in intestinal and pulmonary mucosae and in the dermis of the skin. They increase in number when the body is invaded by some parasites and during allergy attacks. Their granular contents are released to respond to the parasites and allergens. Eosinophil granular content react with products from basophils, mast cells, lymphocytes. For example, eosinophil granules contain histaminase which inactivates histamine; the net effect of released factors is to decrease the inflammatory response and reduce granulocyte migration into the site of invasion.