Hematology 425 Leukopoiesis Russ Morrison October 11, 2006 9/17/2018

Leukopoiesis Leukopoiesis is the development of WBCs WBC development (except lymphycytes) occurs in the same locations as RBCs (review figure 6-1) In WBCs the maturation changes are more unidirectional since with the exception of neoplasia or myeloid metaplasia, the spleen and liver do not participate in WBC formation after birth 9/17/2018

Leukopoiesis Though one term, erythron, is used to define RBC production, there is no corollary for WBC production WBC production involves complex populations of cells with different compartments that they occupy during their life cycle Control mechanisms of cellular behavior are more complex in WBCs than in RBCs 9/17/2018

Leukopoiesis WBC control mechanisms include interrelations with adipose tissue, fibroblasts and endothelial cells Again, as with RBCs, cellular production takes place in all marrow space at birth and by the end of adolescence is found only in the marrow of the proximal ends of the long bones and in flat bones such as the skull and sternum. (review fig.6-2) The inactive marrow of adolescence and adulthood can revert to active marrow in times of stress 9/17/2018

Leukopoiesis WBCs can be divided into categories based on specific function, site of origin or morphology All WBCs exist to defend the body against “nonself” agents This defense is accomplished through intricate cooperation and communication among cells 9/17/2018

Leukopoiesis As a for instance, phagocytes attack and destroy a wide variety of invading matter on their own. However, lymphocytes direct and amplify phagocytic action through the release of lymphokines (a sort of bioresponse mediator) WBCs are divided into granulocytes and lymphocytes based on differentiation at the primitive stem cell level (fig. 11-1) 9/17/2018

Leukopoiesis Lymphocytes are produced in both bone marrow and lymphoid tissue Environmental and hormonal stimuli of lymphocytes are different than those that control granulocytes and monocytes Granulocytes (PMNs) function as destroyers of pyogenic bacteria, monocyte/macrophages are less descriminating in their dietary preferences 9/17/2018

Leukopoiesis Granulocytes contain visible granules and develop in the bone marrow Granulocytes are subdivided according to morphology and according to size/visibility of granules Cells containing large, visible granules are called granulocytes and are further divided into PMNs, Eos and Basos based on differential staining of the granules with Romanowsky-based stain Monocytes contain tiny granules that cause their cytoplasm to appear grainy with light microscopy 9/17/2018

Leukopoiesis Microscopic evaluation of WBCs is the basis of clinical study Flow cytometry of receptor sites, antigenic labeling and even functional studies now contribute to clinical information gathered in the diagnosis and management of disease Cell markers have been given alphanumerical codes (CD1) in which CD stands for cluster designation, discussed in chapter on flow cytometry 9/17/2018

Leukopoiesis, Granulocytes Found in high concentrations in 4 locations called granulocyte pools Bone marrow PB circulation Marginating up against the endothelium of blood vessels Tissues 9/17/2018

Leukopoiesis, Granulocytes Bone marrow pool is large and has 3 functions Proliferation Maturation Storage Cells found in the proliferating component are myeloblasts, promyelocytes and myelocytes, all capable of mitotic division 9/17/2018

Leukopoiesis, Granulocytes The maturation component of the BM consists of metamyelocytes and band forms no longer capable of mitosis but not yet fully functional The storage component of the BM consists of bands and PMNs and holds 25x as many cells as in the circulating PB 9/17/2018

Leukopoiesis, Granulocytes Fully mature granulocytes are stimulated by chemotactic factors and leave the marrow entering the PB where they become part of either the marginating pool of the circulating pool The marginating pool consists of 50% of total PB granulocyte levels where the cells have adhered to blood vessel endothelium or are engaged in diapedesis (egressing into tissue through vessel walls) 9/17/2018

Leukopoiesis, Granulocytes The circulating pool contains the remaining 50% of PB granulocytes and are the cells seen and counted in PB hematologic studies Granulocytes move freely between marginating and circulating pools in a bi-directional flow for a variety of reasons 9/17/2018

Leukopoiesis, Granulocytes Maturation of the Granulocytic Series Begins with the pluripotential stem cell (PSC) PSC commits its progeny to lymphoid or bone marrow origin, for reasons unknown, through the action of growth factors that are either tyrosine kinase receptors or cytokines 9/17/2018

Leukopoiesis, Granulocytes For granulopoiesis, the PSC undergoes stimulation, mitosis and maturation into a stem cell that is specific for bone marrow-derived or myeloid cells This CFU-GEMM matures into another stem cell called the CFU-GM The CFU-GM matures into the earliest cell of the neutrophilic series, the myeloblast 9/17/2018

Leukopoiesis, Granulocytes Cell numbers and function is controlled by complex interaction of humoral factors such as interleukins and CSFs CSFs are categorized by the type of cell stimulated GM-CSF – granulocytes & monocytes/ macrophages G-CSF – granulocytes M-CSF - monocytes/macrophages 9/17/2018

Leukopoiesis, Granulocytes CSF specificity is mediated by receptor sites on precursors and on mature cells Biologic action of receptors consists of a ligand specific low-affinity binding chain and a second, high-affinity chain for binding and signal transduction. The second chain interacts with IL-3 and IL-5 (Chapter 6) 9/17/2018

Neutrophil Maturation - Myeloblast Cells in the BM proliferation pool take 24-48 hours for a single cell cycle Less than 1% of the normal BM compartment is composed of myeloblasts Large, 15-20 um in size Delicate nucleus with prominent nucleoli Small amount of cytoplasm with rough endoplasmic reticulum, a developing Golgi apparatus and an increasing number of azurophilic granules 9/17/2018

Neutrophil Maturation - Myeloblast Cytochemical staining shows presence of myeloperoxidase which is required for intracellular kills Killing function is the first to be operational in the neutrophil cell line Myeloblast is incapable of motility, adhesion and phagocytosis and is therefore nonfunctional 9/17/2018

Neutrophil Maturation - Promyelocyte After a few days in the blast stage, the cell becomes a promyelocyte 1-5% of BM compartment composed of promyelocytes Size is variable and may exceed 20 um, so may be larger than myeloblast Nuclear chromatin may be delicate or may show slight clumping Nuceloli begin to fade 9/17/2018

Neutrophil Maturation - Promyelocyte Granules are present throughout the cytoplasm and on top of the nucleus Motility may develop by the end of this stage Myeloperoxidase is found throughout the cell which with other enzymes can provide the peroxidase/superoxide burst capable of intracellular kill 9/17/2018

Neutrophil Maturation - Myelocyte Production and accumulation of neutrophilic granules is characteristic of the myelocyte The myelocyte is the last cell of the BM compartment capable of mitosis Myelocytes demonstrate morphologic variability as this development stage lasts from 4-5 days and cause alterations in the staining characteristics of the cell 9/17/2018

Neutrophil Maturation - Myelocyte Smaller in size than the promyelocyte (12-18 um) Less than 10% of BM compartment is made up of myelocytes Nucleus is round to oval with a flattened side near the now well-developed Golgi apparatus Nuclear chromatin shows clumping Nucleoli no longer visible 9/17/2018

Neutrophil Maturation - Myelocyte Secondary granules stain pink causing a “dawn of neutrophilia” or pink blush within the cytoplasm Compounds such as alkaline phosphatase begin to concentrate in the cell The cell acquires some motility 9/17/2018

Neutrophilic Maturation - Metamyelocyte The myelocyte becomes a metamyelocyte with the cessation of all DNA synthesis Delineator of maturation change is that the nucleus of the metamyelocyte becomes indented with clumped chromatin Complete collection of primary and secondary granules used to kill and degrade toxic, infectious or non-self agents Cell is not yet capable of responding to chemotactic factors or of initiating phagocytosis 9/17/2018

Neutrophilic Maturation - Metamyelocyte 13-22 % of BM compartment 10-15 um in size Not seen in normal PB Not fully functional, part of the maturation component of the marrow 9/17/2018

Neutrophilic Maturation - Band The band is a transitional form that exists in both the PB and the BM and considered part of both the maturation and storage pools Up to 40% of the WBCs of the BM are bands Represents the “almost mature” neutrophil having full motility, active adhesion properties, and some phagocytic ability 9/17/2018

Neutrophilic Maturation - Band Band forms begin to produce tertiary granules Membrane maturity shows changes in cytoskeleton, surface charge and presence of receptors for complement Once entered into the PB, account for less than 6% of circulating WBCs 10-15 um in size Found in marginating and circulating poos of the PB 9/17/2018

Neutrophilic Maturation - PMN This cell’s nucleus continues to indent until thin strands of membrane and heterochromatin form into segments, hence it is also called a “seg” Polymorphonuclear means “many-shaped nucleus”, describing the varied nuclear shapes Cell is completely functional and spend time in the storage pool of the BM as well as marginating and circulating pools of the PB 50-70% of circulating WBCs of PB 9/17/2018

Neutrophilic Maturation - PMN PMNs spend their life performing phagocytosis and pinocytosis Phagocytosis involves larger material and can be observed with light microscopy, pinocytosis involves small material (liquids) and is observed with EM Both of these function can be performed in the circulation of the blood stream or in the tissues 9/17/2018

Eosinophil Maturation Close relative of the PMN whose secondary granules stain orange-red with Romanowsky-based stains Development of PSCs into eosinophils requires IL-3, IL-5 and GM-CSF and is inhibited by the presence of interferon CFU-GEMM to CFU-Eo to myeloblast Myeloblast to promyelocyte which is indistinguishable from other promyelocytes 9/17/2018

Eosinophil Maturation Myelocyte becomes distinguishable from neutrophilic line due to presence of large, round granules containing major basic protein, which in turn is responsible for the staining qualities of the eosiniphilic granules. Eosinophils spend less than 1 week in the PB Large storage capacity of Eos in BM allow rapid deployment, on demand 9/17/2018

Eosinophil Maturation When stimulated, Eos leave the marrow and pass quickly into the tissues Actively motile, using same migration paths as neutrophils Short transit times in PB cause variability in Eo numbers in the WBC differential Less than 5% of circulating WBCs Allergic response may increase numbers of Eos 9/17/2018

Eosinophil Maturation Mature Eos may be in band form or bilobed while nuclei with higher lobe counts are seldom seen Slightly larger than PMN at 12-17 um 9/17/2018

Basophil Maturation Characterized by presence of large, purple granules Granules are irregularly shaped, unevely distributed and deep purple to black when stained with Romanowsky stains Maturation from stem cell to mature Baso is not well defined, but thought to parallel that of the Eo 9/17/2018

Basophil Maturation As with Eos, Basos can be classifed as myelocytes, metamyelocytes, bands and PMN cells on the basis of nuclear development As with Eos, mature cells with more than 2 nuclear lobes are not usually seen The least common cell in the PB, at less than 1% of circulating WBCs Have high-affinity receptors for the Fc region of IgE 9/17/2018

Monocyte/Macrophage Maturation Monocyte/Macrophage cells mature from monoblast to promonocyte to blood monocyte to free and fixed macrophages, but the mechanism of commitment is not well understood. Granular content vary considerably with more than 50 secretory compounds having been dentified. PB monocytes demonstrate morphologic variability Aggressive motility and adherence may distort the monocytes during PB smear preparation 9/17/2018

Monocyte/Macrophage Maturation Monocyte nucleus is indented or curved with chromatin that is lacy with small clumps Typically the largest cell in the PB Cytoplasm is filled with minute granules that produce a cloudy appearance Cytoplasmic membrane may be irregular, pseudopods and phagocytic vacuoles may be evident 9/17/2018

Monocyte/Macrophage Maturation Described as a transitional cell because it leaves the BM to enter the PB and then leaves to enter tissues in response to chemotactic factors Makes up les than 15% of PB WBC differential Highly motile and tend to marginate along vessel walls with a strong tendency to adhere to surfaces May be stimulated to undergo diapedesis and become free macrophages with increased phagocytic activity 9/17/2018

Monocyte/Macrophage Maturation Macrophages are large, acively phagocytic cells with a size of 15-85 um Pleomorphic in shape, frequently with pseudopods Function is phagocytosis Material ingested is highly variable Pinocytosis also occurs with items less than 2 um in size 9/17/2018

Monocyte/Macrophage Maturation Multistep process of recognition/ attachment, ingestion, intracellular kill, digestion/degradation, and exocytosis occurs in both phagocytosis and pinocytosis. Monocytes kill any recognizable non-self agents including dead or dying cells, bacteria, fungi and viruses. Play a role in processing antigens for lymphocyte recognition and stimulation of lymphocyte transformation. 9/17/2018

Monocyte/Macrophage Maturation May function as anti-tumor agents by phagocytic action of nonself cells via elaboration of tumor necrosis factor and stimulation of lymphocyte activity Macrophages are in 2 categories Free – found in varying concentrations in all sites of inflammation and repair, alveolar spaces and peritoneal and synovial fluids 9/17/2018

Monocyte/Macrophage Maturation Fixed – found in specific concentrations in specific sites such as the nervous system (microglial cells), liver (Kupffer cells), spleen, bone marrow and lymph nodes Macrophages are large, 15-80 um, have ample cytoplasm filled with granules and often have multiple vacuoles Nucleus is round to reniform and may contain 1 or 2 nucleoli 9/17/2018

Lymphocytes The only human WBCs whose site of development is not just BM, but also tisues referred to as primary and secondary lymphoid organs In humans, the primary lymphoid organs are the thymus and bone marrow, the secondary organs include the spleen, Peyer’s patches of the GI tract, the Waldermyer ring of the tonsils and adenoids, the lymph nodes and modules scattered throughout the body 9/17/2018

Lymphocytes Lymphocytes circulate throughout the body in both PB and lymph which act as carrier streams to bring the lymphocytes to sites of activity Lymphocytes migrate from thoracic duct through vessel endothelium to lymph nodes to blood stream and back. Lymphocytes are categorized in a variety of ways and may be short-lived or long-lived cells Lymphocytes may produce antibodies or lymphokines and have different surface charges, densities and antigen receptors. 9/17/2018

Lymphocytes - Development The PSC results in a stem cell for the lymphoid cell (CFU-L) as a result of hormonal stimuli The CFU-L matures in several environments Thymus and BM give rise to lymphocytes, foster differentiation and are indepentendent of antigenic stimulation 9/17/2018

Lymphocytes - Development Cells that develop under the influence of the thymus are called T cells and have specific receptors and responses. B cells develop from the BM and have a different set of functions and receptors. The end cell of the B lymphocyte maturation is the plasma cell Once the environmental effects of the thymus and BM have been achieved, lymphocytes migrate to secondary lymphatic tissues such as the spleen and tonsils, which act as the main repositories for already differentiated lymphocytes. 9/17/2018

Lymphocytes - Development Cellular interactions for the presentation of antigen to the cells have a critical role in priming cells for proliferation and impact cell maturation, especially T cells. Once primed, the cells are now responsive to specific antigens. Lymphocytes demonstrate lymphoblast, prolymphocyte and mature lymphocyte stages when stained with Romanowsky stains. 9/17/2018

Lymphocytes - Development Lymphocyte % in the PB varies, depending on age. Children under the age of 4 have a higher proportion of lymphocytes in the PB than do adults Lymphocytes are the second most common WBC of the PB making up 20-40% of WBCs. 20-35% of circulating lymphocytes are B cells 9/17/2018

Lymphocytes - Maturation Lymphoblast to prolymphocyte Lymphoblast is small, 10-18 um Round to oval nucleus Loose chromatin with one or more active nucleoli Scanty cytoplasm Prolymphocyte difficult to distinguish, subtle changes, more clumped chromatin, lessening nucleolar priminence, change in thickness of the nuclear membrane 9/17/2018

Lymphocytes - Maturation Prolymphocyte to Lymphocyte Lymphocytes vary mostly by size Small – 9 um in diameter, non-dividing or resting Medium – 11-14 um, non-dividing Large – 15 um, more generous cytoplasm that is deep blue when stained Morphologic variants (table 11-2) 9/17/2018

Lymphocytes – Immunologic Differentiation Lymphocytes may be classified by immunologic function B Cells Possess cytoplasmic IG concentrations of IgD and IgM Some membrane receptors are apparent The fully committed B lymphocyte is the plasma cell Demonstrate class I and class II human leukocyte antigens (HLA-A, HLA-B, HLA-C and HLA-D, HLA-DR) 9/17/2018

Lymphocytes – Immunologic Differentiation T Cells The primitive T cell, CFU-L, travels to the thymus Acquires a transferrin receptor that is specific to proliferation Mature T cells lose all precursor markers and have an active helper or suppressor function T cells are further differentiated through presence or absence of HLA-D antigens T cells possess HLA-A, HLA-B and HLA-C class I antigens 9/17/2018

Lymphocytes - Activity The main function of the lymphocyte is to regulate immune function If foreign material is completely engulfed, degraded and disposed of by phagocytes, no immune response occurs If digestion is incomplete, antigenic fragments are transported to lymph nodes In the lymph node the antigen is fixed to the exterior surface and brought into the lysozymes of the macrophage 9/17/2018

Lymphocytes - Activity The antigen is processed and once that occurs, proliferation occurs Development of clones of antigen-specific B lymphocytes and cytotoxic T cells begins Activity that accompanies clonal expansion required for antigen removal can be seen in the morphology of cells called reactive lymphocytes 9/17/2018