STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE “The WBCs”

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE The basic function of blood leukocytes is as a defense system against infectious foreign invaders and non-infectious challenge. This is divided into two separate, but interconnected events: Phagocytosis Involves granulocytes and monocytes Development of a specific immune response Involves monocytes (macrophages) and lymphocytes The vascular system is only a temporary residence for leukocytes. The main function of the vasculature with respect to WBCs is to transport the leukocytes to body tissues.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Leukocytes are differentiated from each other by nuclear and cytoplasmic characteristics: Granulocytes (contain granules) The granules in eosinophils have affinity for the acid part of the Wright’s stain and they stain orange-pink The granules of basophils have affinity for the basic part of the stain and they stain bluish-black. The granules of neutrophils have an affinity for both acid and basic parts of the stain and they stain pinkish-blue.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Agranulocytes (lack granules): Monocytes are usually large with a horseshoe shaped nucleus Lymphocytes may be small (non-reactive) with a large nuclear to cytoplasmic ratio or large (reactive) with a smaller nuclear to cytoplasmic ratio. The nucleus is usually round (small lymphs) or may be slightly indented (large lymphs). Leukocytes differentiate into mature cells from the pluripotential stem cell in the bone marrow.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Normally only mature cells are released into the peripheral blood. Mature cells may also remain as storage in the bone marrow. At birth the leukocyte count is high: 9-30 x 109/L ; in adults the normal is 4-10.5 x 109/L An increase or decrease in WBC count may be caused by an alteration of all WBC cell lines, but more commonly results from an alteration of only one type of WBC.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Therefore, a differential is important. From the differential and the WBC count, the absolute values for each type of WBC can be calculated (relative differential (%) x total WBC count). Most variations in the leukocyte count are due to increases or decreases in the number of neutrophils since by percentage they are the most numerous:

Age and Race-specific Reference Ranges for Leukocyte Count and Differential Birth 6 Months 4 Years Adult Adult of African Descent Total leukocyte count (x109/L) 9.0-30.0 6.0-18.0 4.5-13.5 4.0-11.0 3.0-9.0 Segmented neutrophil (%) 50-60 25-35 35-45 40-80 45-55 Absolute (x109/L) 4.5-18.0 1.5-6.3 1.5-8.5 2.0-6.8 1.5-5.0 Band neutrophil (%) 5-14 0-5 .5-4.2 0-1.0 0-0.7 0-0.5 Lymphocyte (%) 55-65 50-65 2.0-11.0 4-13.5 2.0-8.8 1.0-4.0 Monocyte (%) 2-10 0.2-3.0 0.1-2.0 0.1-1.4 0.2-1.0 Eosinophil (%) 0-1.5 0-0.9 Basophil (%) 0-2 0-0.6 0-0.4 0-0.3 0-0.2

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Neutrophil maturation- As the neutrophil matures: Size decreases Nucleoli disappear Chromatin condenses, indents, and finally segments The cytoplasm goes from an agranular, basophilic (blue) to a granular, more acidic (pink) color The order of maturation from committed stem cell to the mature neutrophil (also called a polymorphonuclear leukocyte or PMN cell) is:

MYELOBLAST Myeloblast – has a high N:C ration, fine,lacey chromatin, visible nucleoli, and basophilic cytoplasm:

PROMYELOCYTE Promyelocyte – basophilic staining primary granules containing esterase, lysozyme, serprocidins , defensins, bacterial permeability increasing protein, and myeloperoxidase are formed, nucleoli are indistinct and chromatin condenses:

MYELOCYTE Myelocyte –secondary, neutrophilic or specific granules containing lactoferrin, histaminase, cathelicidins, lysozyme and collagenase now appear and the concentration of the primary granules decreases. Secretory vesicles containing alkaline phosphatase, complement receptor 1, and cytochrome b are also made:

METAMYELOCYTE Metamyelocyte – nuclear indentation begins:

BAND Band – also known as a stab. Has a horseshoe shaped nucleus. Normal peripheral blood may contain 1-10% bands (depends upon criteria for calling a cell a band):

VARIOUS STAGES OF NEUTROPHIL MATURATION Mature segmented neutrophil – may have 2-5 lobes:

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Neutrophil kinetics The absolute value is 2-7 x 109/L The absolute count is diurnal, i.e., it is higher in the evening Alterations in the peripheral blood concentration of neutrophils are often the first indication of an underlying pathology Neutropenia is when the concentration is < 2 x109/L and when this occurs it can lead to infections Neutrophilia is when the concentration is > 7 x109/L and this occurs as a response to bacterial infection, metabolic or drug intoxication, or tissue necrosis.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Only about half of the peripheral neutrophils are freely circulating in the blood, while the other half are attached to the vessel walls (called the marginating pool). There is a free equilibrium between the circulating and marginating pool. Upon antigenic stimulation, the marginating neutrophils will move into the tissues (diapedesis). The average neutrophil circulates~ 10 hours before diapedesis.

MOVEMENT OF PMNS TO TISSUES

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE There are two pools of neutrophils in the bone marrow: Mitotic and capable of DNA synthesis (myeloblasts up to myelocytes – takes 3-6 days to mature to this stage) Post-mitotic – serve as a storage pool of neutrophils (metamyelocytes up the mature segmented neutrophils – takes 5-7 days to mature to this stage). These pools in the bone marrow represents 15-20 times the number of circulating neutrophils!

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Under conditions of stress the normal marrow transit time can be increased by: Accelerated maturation Skipping cellular divisions Releasing cells early from the bone marrow Normally the input from the bone marrow is equal to the output to the tissues – this is achieved by feedback loops.

NEUTROPHIL DISTRIBUTION

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Neutrophil function – The characteristic response of the blood leukocyte count to bacterial infections is neutrophilia with an increase in the % of bands and even some metamyelocytes in the peripheral blood. This is called a shift to the left. Neutropenia may be a bad prognostic sign (only if it is due to depletion of the bone marrow stores rather than to a transient increase in marginating neutrophils).

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Neutrophil morphology may also be altered during infections Toxic granulation (cytoplasmic, primary granules become prominent) Dohle bodies may be seen – are large bluish bodies consisting of aggregated RER The cytoplasm may become vacuolated May see ingested microorganisms

TOXIC GRANULATION

DOHLE BODIES

VACUOLES

NEUTROPHIL RESPONSE TO INFECTION

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE To participate in an inflammatory reaction, which is the body’s response to infection, neutrophils enter the tissues where they are attracted by chemotactic stimuli released after tissue injury and during an inflammatory response. In the tissues, the neutrophils participate in phagocytosis (opsonization helps). Upon phagocytosis of the microorganism into a phagocytic vacuole, a plasma membrane bound oxidase is activated (oxidative or respiratory burst) resulting in the formation of oxygen by-products that may be toxic to microorganisms (superoxide radical, hydrogen peroxide, etc.)

PMN FUNCTION

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Next primary and specific granules and secretory vesicles (collectively called lysosomes) fuse with the phagocytic vacuole, releasing their contents (degranulation). The enzymes and antimicrobial peptides such as defensins, serprocidins , and bacterial permeability increasing protein (BPI) present in the granules may act to kill the ingested microorganisms. In this process most neutrophils die and are themselves phagocytosed by macrophages.

PHAGOCYTOSIS

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Eosinophils Have a maturation similar to that of neutrophils, but only one type of granule is formed and it contains acid phosphotase, peroxidases, ribonuclease, etc. Eosinophils function in phagocytosis and killing of bacteria, but they are more sluggish and less efficient than neutrophils. Their primary function is against helminth parasites. Eosinophilia may occur in allergic conditions and with parasitic infections, and with chronic inflammation.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Basophils Basophils also have a similar maturation process, but their metachromatic granules contain histamine and heparin. Basophils share a common hematopoietic stem cell with tissue mast cells and functionally the two are similar. They are mediators of the inflammatory response, particularly in hypersensitivity reactions.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Both have membrane receptors for the Fc region of IgE. Upon attachment of antigen to the IgE, degranulation and release of mediators of hypersensitivity reactions occur. This leads to vasodilation, increased vascular permeability, smooth muscle contractions, and increased secretions. Monocyte maturation Monocytes are derived from a committed bipotential stem cell capable of maturing into a monocyte or a neutrophil. The bipotential stem cell matures into a monoblast containing an agranular cytoplasm. It can be differentiated from a myeloblast by cytochemical stains.

MONOBLAST

PROMONOCYTE

MATURE MONOCYTE

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE When mature monocytes leave the blood and enter the tissues, they mature into macrophages. This is accompanied by progressive enlargement of the cell. The cells can live for months in the tissues and are known collectively as histiocytes. There is no reserve pool of monocytes in the bone marrow. All are directly released into the peripheral circulation where they circulate 12-14 hours before diapedesis occurs and they enter the tissues. The total vascular pool = the freely circulating monocytes and the marginating monocytes which is three times the number of freely circulating monocytes.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Monocyte and macrophage function They function in phagocytosis of microorganisms and cellular debris They secrete substances (cytokines) that affect the function of other cells, especially lymphocytes. They are important in the processing of and presentation of antigens to lymphocytes for activation and differentiation. Lymphocytes – their primary function is to react with antigens and to work with macrophages in modulating (controlling) the immune response.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Lymphocyte maturation From the pluripotential stem cell comes the committed lymphoid stem cell which will eventually differentiate into B and T lymphocytes. This lymphopoiesis is divided into two phases Antigen independent phase – this occurs in the primary lymphoid tissues (bone marrow, thymus, fetal liver, and yolk sac) and results in the formation of immunocompetent B and T cells In the bone marrow the plupotential stem cell differentiates into a committed lymphoid stem cell.

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Those that will become T cells migrate to the thymus for differentiation and maturation Those that will become B cells remain in the bone marrow for differentiation and maturation In those two environments the committed stem cell matures into a lymphoblast The lymphoblast has a high N:C ratio, like the myeloblast, but it is much smaller. The lymphoblast becomes a prolymphocyte The prolymphocyte becomes an immunocompetent T (those that mature in the thymus) or B (those that mature in the bone marrow) cell. Immunocompetent B and T cells have acquired receptors (antibodies for B cells and T cell receptor for T cells) that recognize and bind a specific a specific antigen. One cannot tell the difference between a B or T cell under the microscope.

AG INDEPENDENT MATURATION - LYMPHOBLAST

AG INDEPENDENT MATURATION - PROLYMPHOCYTE

AG INDEPENDENT MATURATION – IMMUNOCOMPETENT LYMPHOCYTE

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE The mature lymphocyte has extreme size variability which is primarily dependent upon the amount of cytoplasm. The majority are small lymphocytes. Large lymphocytes, which probably represent activated lymphocytes (see below), are often mistaken for monocytes. When compared to a monocyte, the nuclear chromatin of a lymphocyte is more condensed (clumpy) and the cytoplasm is more clear and non-granular. The antigen dependent phase of maturation begins when a specific antigen binds to the antigen receptor on the surface of an immunocompetent B or T cell. Binding triggers a sequence of events called blast transformation: The immunocompetent cell becomes a reactive lymphocyte (sometimes called Downey cells).

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE The reactive lymphocyte becomes an immunoblast which will undergo clonal expansion to increase the number of lymphocytes that can recognize and bind to the antigen that triggered the whole reaction. For T cells, these cells may then differentiate into memory cells or into effector cells that help to mediate the immune response (T helper, T supressor, cytotoxic T cells, or delayed hypersensitivity T cells). In the case of B cells they may differentiate into memory cells or plasma cells that will secrete antibody of the same specificity that was on the original immunocompetent B cell.

AG DEPENDENT MATURATION – REACTIVE LYMPHOCYTE

AG DEPENDENT MATURATION - IMMUNOBLAST

AG DEPENDENT MATURATION – PLASMOCYTOID LYMPHOCYTE

AG DEPENDENT MATURATION – PLASMA CELL

STRUCTURE AND FUNCTION OF LEUKOPOIETIC TISSUE Lymphocyte kinetics The peripheral blood contains only about 5% of the total body lymphocytes Most of the lymphocytes reside in the lymphoid organs (mainly the spleen and lymph nodes, but also the thymus, and bone marrow)

LYMPHOCYTE MATURATION