Little attention has been given to the evolutionary origin of blood. Very little scientific literature exists on the subject. And remarkably: Understanding how the blood system is formed is an ongoing fundamental research challenge, even in 2015. Hematopoiesis is the description of how blood cellular components form. Blood and the developing blood cells and their precursors are produced in the bone marrow through pluripotential Hematopoietic stem cells (HSCs) which give rise to all ten different type of blood cells through the process of haematopoiesis These blood cells are: macrophages , neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, platelets, T cells, B cells, and natural killer cells.
Question: At least six of the different cell types in blood are life essential. If one is missing, life ceases to exist. Had these blood types not to emerge together, to make the life of a multicellular organism possible?
Most blood types require intermediate steps in their development until becoming final cell types used in blood. These intermediate cell types have no function in the organism. Why would evolution produce them?
Bruce Alberts and his colleagues noted: “Blood contains many types of cells with very different functions, ranging from the transport of oxygen to the production of antibodies. Some of these cells function entirely within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere (1994, p. 1161).
The process of developing the diverse blood cell repertoire from stem and progenitor cells termed hematopoiesis has been subject to considerable investigation. However, key steps in the complex process of hematopoiesis, including hematopoietic stem cell generation during embryogenesis, hematopoietic stem, and progenitor cell expansion remain incompletely understood.
Furthermore: Red blood cells, most white blood cells, and platelets are produced in the bone marrow, the soft fatty tissue inside bone cavities.
And bone formation is an irreducible complex, extremely complex process:
Natural killer cells are a type of immune cell that has granules (small particles) with enzymes that can kill tumor cells or cells infected with a virus. A natural killer cell is a type of white blood cell. Also called NK cell and NK-LGL. They are part of the innate immune defense against infection and cancer, and are especially useful in combating certain viral pathogens9
T cells are essential for human immunity. 8
B cells are a type of white blood cell that makes antibodies. B lymphocytes are part of the immune system and develop from stem cells in the bone marrow. Primary B-cell immunodeficiencies (B-PID) constitute a heterogeneous group of immunodeficiencies characterized by defective production of antigen-specific antibodies and predisposition to recurrent and severe infections 9
Dendritic cells are needed to control B and T lymphocytesm, and capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses.3
Macrophages are essential components of the innate immune system. They are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the types of proteins specific of healthy body cells on its surface in a process called phagocytosis.2
Neutrophils are a required type of immune cell that is one of the first cell types to travel to the site of an infection. Neutrophils help fight infection by ingesting microorganisms and releasing enzymes that kill the microorganisms. A neutrophil is a type of white blood cell,
Basophils contain anticoagulant heparin, which prevents blood from clotting too quickly. They also contain the vasodilator histamine, which promotes blood flow to tissues.
Eosinophils effector functions include production of: cationic granule proteins and their release by degranulation, the production of reactive oxygen species such as hypobromite, superoxide, and peroxide, production of lipid mediators like the eicosanoids, enzymes, such as elastase. growth factors such as TGF beta, VEGF, and PDGF. etc....
Red blood cells ( erythrocytes ) are the most common type of blood cell and essential for the vertebrate organism's delivering oxygen (O2) to the body tissues—via blood flow through the circulatory system 6
Platelets are a essential component of blood whose function (along with the coagulation factors) is to stop bleeding by clumping and clotting blood vessel injuries 7
Red Blood Cells: Centerpiece in the Evolution of the Vertebrate Circulatory System
Blood is a bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells. In vertebrates, it is composed of blood cells suspended in blood plasma. Plasma, which constitutes 55% of blood fluid, is mostly water, and contains dissipated proteins, glucose, mineral ions, hormones, carbon dioxide (plasma being the main medium for excretory product transportation), and blood cells themselves. Albumin is the main protein in plasma, and it functions to regulate the colloidal osmotic pressure of blood. According to H.Glicksman, the regulation of blood pressure requires a irreducible complex system .
The blood cells are mainly red blood cells, white blood cells and platelets. The most abundant cells in vertebrate blood are red blood cells. These contain hemoglobin, an iron-containing protein, which facilitates oxygen transport by reversibly binding to this respiratory gas and greatly increasing its solubility in blood. In contrast, carbon dioxide is almost entirely transported extracellularly dissolved in plasma as bicarbonate ion.
The bone marrow, which is the flexible tissue in the interior of bones, forms a suitable environment for stem cell survival, growth and development. It is composed of stromal cells and a microvascular network. Bones are an amazing example of design, present in all vertebrates. They have a huge advantage over man-made girders, in that they are constantly rebuilding and redesigning themselves to cope with changing stress directions.12 This involves a fine balance of the activity of bone-depositing cells (osteoblasts) and bone resorbing cells (osteoclasts). It’s been recently shown that thyroid-stimulating hormone (TSH), best known for what its name says—stimulating the production of hormones in the thyroid gland—has an important role. It oversees both types of cells—without it, bones have osteoporosis in some parts (too little bone, so very weak), and are too dense in other patches. So both are essential. For bone formation, over 24 different proteins, cells, Vitamins, transcription factors etc. are required. So, we can confidently say, the production of blood requires a hudge number of different body parts, which are essential. Blood by its own has no function. The vascular system without blood has no function. Bones require a vascular system, which by their own are required to make blood. The vertebrate body is a hudge irreducible complex sytem, that could not have emerged gradually. Each multicellular organism had to emerge fully, in its interdependent form.
Hematopoiesis. The mystery of blood Cell and vascular formation
OVERVIEW OF BLOOD
Little attention has been given to the evolutionary basis of blood. Here, i will employ a description of what blood consists of, hematopoiesis ( how blood cellular components form ), function and essentiality of each blood component in health and pathological consequences through desease or a component inexistence, and elucidating if a gradual evolutionary step by step pathway is hypothetically and theoretically possible, or if the origin of blood is better explained through intelligent design and sudden creation.
Blood is a fluid connective tissue that circulates through the cardiovascular system. Like the other connective tissues, blood consists of cells and an extracellular component. Total blood volume in the average adult is about 6 L or 7% to 8% of total body weight. The heart’s pumping action propels blood through the cardiovascular system to the body tissues.
Blood’s many functions include:
• delivery of nutrients and oxygen directly or indirectly to
• transport of wastes and carbon dioxide away from cells,
• delivery of hormones and other regulatory substances to
and from cells and tissues,
• maintenance of homeostasis by acting as a buffer and
participating in coagulation and thermoregulation, and
• transport of humoral agents and cells of the immune
system that protect the body from pathogenic agents,
foreign proteins, and transformed cells (i.e., cancer cells).
Blood consists of cells and their derivatives and a protein-rich fluid called plasma. Blood cells and their derivatives include:
• erythrocytes, also called red blood cells (RBCs);
• leukocytes, also known as white blood cells (WBCs); and
• thrombocytes, also termed platelets.
Plasma is the liquid extracellular material that imparts fluid properties to blood. The relative volume of cells and plasma in whole blood is approximately 45% and 55%, respectively. The volume of packed erythrocytes in a sample of blood is called the hematocrit (HCT) or packed cell volume (PCV). The hematocrit is measured by centrifuging a blood sample to which anticoagulants have been added, and then calculating the percentage of the centrifuge tube volume occupied by the erythrocytes compared with that of the whole blood (Fig. 10.1).
A normal hematocrit reading is about 39% to 50% in men and 35% to 45% in women; thus, 39% to 50% and 35% to 45% of the blood volume for men and women, respectively, consists of erythrocytes. Low hematocrit values often reflect reduced numbers of circulating erythrocytes (a condition called anemia) and may indicate significant blood loss caused by internal or external bleeding. Leukocytes and platelets constitute only 1% of the blood volume. In a blood sample that has been centrifuged, the cell fraction (the part of the sample that contains the cells) consists mainly of packed erythrocytes (99%). The leukocytes and platelets are contained in a narrow, light-colored layer between the erythrocytes and plasma called the buffy coat (see Fig. 10.1).
As Table 10.1 indicates, there are nearly 1,000 times more erythrocytes (5 10^12 cells/L of blood) than leukocytes (7 10^9/L of blood).
Although the blood cells are the major objects of interest in histology, a brief examination of plasma is also useful. The composition of plasma is summarized in Table 10.2.
More than 90% of plasma by weight is water, which serves as the solvent for a variety of solutes, including proteins, dissolved gases, electrolytes, nutrients, regulatory substances, and waste materials. The solutes in the plasma help maintain homeostasis, a steady state that provides optimal pH and osmolarity for cellular metabolism. Plasma proteins consist primarily of albumin, globulins, and fibrinogen. Albumin is the main protein constituent of the plasma, accounting for approximately half of the total plasma proteins. It is the smallest plasma protein (about 70 kDa) and is made in the liver. Albumin is responsible for exerting the concentration gradient between blood and extracellular tissue fluid. This major osmotic pressure on the blood vessel wall, called the colloid osmotic pressure, maintains the correct proportion of blood to tissue fluid volume. If a significant amount of albumin leaks out of the blood vessels into the loose connective tissue or is lost from the blood to urine in the kidneys, then the colloid osmotic pressure of the blood decreases, and fluid accumulates in the tissues. (This increase in tissue fluid is most readily noted by swelling of the ankles at the end of a day.) Albumin also acts as a carrier protein; it binds and transports hormones (thyroxine), metabolites ( bilirubin), and drugs (barbiturates). Globulins include the immunoglobulins ( γ-globulins), the largest component of the globulin fraction, and nonimmune globulins (α-globulin and β-globulin). The immunoglobulins are antibodies, a class of functional immune-system molecules secreted by plasma cells. Nonimmune globulins are secreted by the liver. They help maintain the osmotic pressure within the vascular system and also serve as carrier proteins for various substances such as copper (by ceruloplasmin), iron (by transferrin), and the protein hemoglobin (by haptoglobin). Nonimmune globulins also include fibronectin, lipoproteins, coagulation factors, and other molecules that may exchange between the blood and the extravascular connective tissue. Fibrinogen, the largest plasma protein (340 kilodaltons), is made in the liver. In a series of cascade reactions with other coagulation factors, soluble fibrinogen is transformed into the insoluble protein fibrin (323 kilodaltons).
During conversion of fibrinogen to fibrin, fibrinogen chains are broken to produce fibrin monomers that rapidly polymerize to form long fibers. These fibers become cross-linked to form an impermeable net at the site of damaged blood vessels, thereby preventing further blood loss. With the exception of these large plasma proteins and regulatory substances, which are small proteins or polypeptides, most plasma constituents are small enough to pass through the blood vessel wall into the extracellular spaces of the adjacent connective tissue. In general, plasma proteins react with common fixatives; they are often retained within the blood vessels in tissue sections. Plasma proteins do not possess a structural form above the molecular level; thus, when they are retained in blood vessels in the tissue block, they appear as a homogeneous substance that stains evenly with eosin in hematoxylin and eosin (H&E)–stained sections.
Serum is the same as blood plasma except that clotting factors have been removed.
For laboratory purposes, samples of blood are often drawn from a vein (the procedure called venipuncture).When blood is removed from the circulation, it immediately clots. A blood clot consists mostly of erythrocytes entangled in a network of fine fibers composed of fibrin. To prevent clotting of a blood sample, an anticoagulant such as citrate or heparin is added to the blood specimen as it is obtained. Citrate binds calcium ions, which are essential for triggering the cascade of coagulation reactions; heparin deactivates the clotting factors in the plasma. Plasma that lacks coagulation factors is called serum.
Erythrocytes are anucleate, biconcave discs.Erythrocytes or red blood cells (RBCs), are anucleatecells devoid of typical organelles. They function only within the bloodstream to bind oxygen for delivery to the tissues and, in exchange, bind carbon dioxide for removal from the tissues. Their shape is that of a biconcave discs with a diameter of 7.8 μm, an edge thickness of 2.6 μm, and a central thickness of 0.8 μm. This shape maximizes the cell’s surface area (140 μm2), an important attribute in gas exchange.
The life span of erythrocytes is approximately 120 days, after which most (90%) of them are phagocytosed by macrophages in the spleen, bone marrow, and liver. The remaining aged erythrocytes (10%) break down intravascularly, releasing insignificant amounts of hemoglobin into the blood. In H&E–stained sections, erythrocytes are usually 7 to 8 μm in diameter. Because their size is relatively consistent in fixed tissue, they can be used to estimate the size of other cells and structures in tissue sections; in this role, erythrocytes are appropriately referred to as the “histologic ruler.” Because both living and preserved erythrocytes usually appear as biconcave discs, they can give the impression that their form is rigid and inelastic (Fig. 10.2).
They are, in fact, extremely deformable. They pass easily through the narrowest capillaries by folding over on themselves. They stain uniformly with eosin. In thin sections viewed with the transmission electron microscope (TEM), the contents of an erythrocyte appear as a dense, finely granular material.
The shape of the erythrocyte is maintained by membrane proteins.
The cell membrane of an erythrocyte is composed of a typical lipid bilayer and contains two functionally significant groups of proteins.
• Integral membrane proteins represent most of the proteins in the lipid bilayer. They consist of two major families: glycophorins and band 3 proteins. The extracellular domains of these integral membrane proteins are glycosylated and express specific blood group antigens. Glycophorin C, a member of the glycophorin family of transmembrane proteins, plays an important role in attaching the underlying cytoskeletal protein network to the cell membrane. Band 3 protein binds hemoglobin and acts as an additional anchoring site for the cytoskeletal proteins (Fig. 10.3).
• Peripheral membrane proteins reside on the inner surface of the cell membrane. They are organized into a two-dimensional hexagonal lattice network that laminates the inner layer of the membrane. The lattice itself, which is positioned parallel to the membrane, is composed mainly of cytoskeletal proteins including spectrin tetramers, actin, band 4.1 protein, adducin, band 4.9 protein, and tropomyosin (see Fig. 10.3) that form a network or mesh. The lattice is anchored to the lipid bilayer by the globular protein ankyrin, which interacts with band 4.2 protein as well as with band 3 integral membrane protein.
This unique cytoskeletal arrangement contributes to the shape of the erythrocyte and imparts elastic properties and stability to the membrane. The cytoskeleton is not static; it undergoes continuous rearrangement in response to various physical factors and chemical stimuli as the cell moves through the vascular network.
Any defect in the expression of genes that encode these cytoskeleton proteins can result in abnormally shaped and fragile erythrocytes. For instance, hereditary spherocytosis is caused by a primary defect in spectrin gene expression that results in spherical erythrocytes. Hereditary elliptocytosis is caused by a deficiency in band 4.1 proteins that results in elliptical erythrocytes. In
both conditions, erythrocytes are unable to adapt to changes in their environment (e.g., osmotic pressure and mechanical deformations), which results in premature destruction of the cells, or hemolysis.
Erythrocytes contain hemoglobin, a protein specialized for the transport of oxygen and carbon dioxide.
Erythrocytes transport oxygen and carbon dioxide bound to the protein hemoglobin (68 kilodaltons). A monomer of hemoglobin is similar in composition and structure to myoglobin, the oxygen-binding protein found in striated muscle. A high concentration of hemoglobin is present within erythrocytes and is responsible for their uniform staining with eosin and the cytoplasmic granularity seen with the TEM. The disc shape of the erythrocyte facilitates gas exchange because more hemoglobin molecules are closer to the plasma membrane than they would be in a spherical cell. Thus, gases have less distance to diffuse within the cell to reach a binding site on the hemoglobin. Hemoglobin consists of four polypeptide chains of globin, each complexed to an iron-containing heme alpha, beta, gamma, and sigma group (Fig. 10.4). The structure of the polypeptide chains varies.
Leukocytes are subclassified into two general groups. The basis for this division is the presence or absence of prominent specific granules in the cytoplasm. Cells containing specific granules are classified as granulocytes (neutrophils, eosinophils, and basophils), and cells that lack specific granules are classified as agranulocytes (lymphocytes and monocytes). However, both agranulocytes and granulocytes possess small number of nonspecific
azurophilic granules, which are lysosomes. The relative number of the various leukocytes is given in Table 10.1.
The function of blood vessels is to carry blood. Blood contains many types of cells, with functions that range from the transport of oxygen to the production of antibodies. Some of these cells stay within the vascular system, while others use the vascular system only as a means of transport and perform their function elsewhere. All blood cells, however, have certain similarities in their life history. They all have limited life-spans and are produced throughout the life of the animal. Most remarkably, they are all generated ultimately from a common stem cell, located (in adult humans) in the bone marrow. This hematopoietic (blood-making) stem cell is thus multipotent, giving rise to all the types of terminally differentiated blood cells as well as some other types of cells, such as the osteoclasts in bone. The hematopoietic system is the most complex of the stem-cell systems in the mammalian body.
Neutrophils are the most numerous WBCs as well as the most common granulocytes
Neutrophils measure 10 to 12 μm in diameter in blood smears and are obviously larger than erythrocytes. Although named for their lack of characteristic cytoplasmic staining, they are also readily identified by their multilobal nucleus; thus, they are also called polymorphonuclear neutrophils or polymorphs. Mature neutrophils possess two to four lobes of nuclear material joined by thinner nuclear strands . The arrangement is not static; rather, in living neutrophils the lobes and connecting strands change their shape, position, and even number. The chromatin of the neutrophil has a characteristic arrangement. Wide regions of heterochromatin are located chiefly at
the periphery of the nucleus in contact with the nuclear envelope. Regions of euchromatin are located primarily at the center of the nucleus with relatively smaller regions contacting the nuclear envelope (Fig. 10.5). In women, the Barr body (the condensed, single, inactive X chromosome) forms a drumstickshaped appendage on one of the nuclear lobes.
Neutrophils contain three types of granules.
The cytoplasm of a neutrophil contains three kinds of granules. The different types of granules reflect the various phagocytotic functions of the cell.
• Specific granules (secondary granules) are the smallest granules and are at least twice as numerous as azurophilic granules. They are barely visible in the light microscope; in electron micrographs, they are ellipsoidal (see Fig. 10.5). Specific granules contain various enzymes (i.e., type IV collagenase, phospholipase) as well as complement activators and other antimicrobial peptides (i.e., lysozymes, lactoferrins).
• Azurophilic granules (primary granules) are larger and less numerous than specific granules. They arise early in granulopoiesis and occur in all granulocytes, as well as in monocytes and lymphocytes. The azurophilic granules are the lysosomes of the neutrophil and contain myeloperoxidase (MPO) (a peroxidase enzyme), which appears as a finely stippled material with the TEM. Myeloperoxidase helps to generate highly reactive bactericidal hypochlorite and chloramines. In addition to containing a variety of the typical acid hydrolases, azurophilic granules also contain cationic proteins called defensins, which function analogously to antibodies and the antimicrobial peptide cathelicidin to kill pathogens.
• Tertiary granules in neutrophils are of two types. One type contains phosphatases (enzymes that remove a phosphate group from a substrate) and is sometimes called a phosphasome. The other type contains metalloproteinases, such as gelatinases and collagenases, which are thought to facilitate the migration of the neutrophil through the connective tissue. Aside from these granules, membrane-bounded organelles
are sparse. A small Golgi apparatus is evident in the center of the cell, and mitochondria are relatively few in number (see Fig. 10.5).
Neutrophils are motile cells; they leave the circulation and migrate to their site of action in the connective tissue.An important property of neutrophils and other leukocytes is their motility. Neutrophils are the most numerous of the first wave of cells to enter an area of tissue damage. Their migration is controlled by the expression of adhesion molecules on the neutrophil surface that interact with corresponding ligands on endothelial cells (Fig. 10.6) and are often involved in cell binding.
The initial phase of neutrophil migration occurs in the postcapillary venules and is regulated by a mechanism involving neutrophil–endothelial cell recognition. Selectins (a type of cell adhesion molecule) on the surface of the circulating neutrophil (CD62L) interact with receptors (GlyCAM-1) on the surface of the endothelial cells. The neutrophil becomes partially tethered to the endothelial cell as a result of this interaction, which slows the neutrophil and causes it to roll on the surface of the endothelium. In the second phase, another group of adhesion molecules on the neutrophil surface, called integrins (VLA-5), are activated by chemokine signals from the endothelial cells. In the third phase, integrins and other adhesion molecules from the immunoglobulin superfamily (e.g., intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) expressed on the neutrophil surface engage with their specific receptors on the endothelial cells, attaching the neutrophil to the endothelial cell. The neutrophil then extends a pseudopod to an intercellular junction. Histamine and heparin released at the injury site by perivascular mast cells open the intercellular junction, allowing the neutrophil to migrate into the connective tissue. With the TEM, the cytoplasmic contents of a neutrophil pseudopod appear as an expanse of finely granular cytoplasmic matrix with no membranous organelles (see Fig. 10.5). The finely granular appearance is attributable to the presence of actin filaments, some microtubules, and glycogen, which are involved in the extension of the cytoplasm to form the pseudopod and the subsequent contraction that pulls the cell forward. Once the neutrophil enters the connective tissue, further migration to the injury site is directed by a process known as chemotaxis, the binding of chemoattractant molecules and extracellular matrix proteins to specific receptors on the surface of the neutrophil.
Neutrophils are active phagocytes that utilize a variety of surface receptors to recognize bacteria and other infectious agents at the site of inflammation
Once at the site of tissue injury, the neutrophil must first recognize any foreign substances before phagocytosis can occur. Like most phagocytic cells, neutrophils have a variety of receptors on their cell membrane that can recognize and bind to bacteria, foreign organisms, and other infectious agents (Fig 10.7).
Some of these organisms and agents bind directly to neutrophils (no modifications of their surfaces are required), whereas others must be opsonized (coated with antibodies or complement) to make them more attractive to the neutrophil. The most common receptors used by neutrophils during phagocytosis include the following.
• Fc receptors on the neutrophil surface bind to the exposed Fc region of IgG antibodies that coat bacterial surfaces (see Fig 10.7). Binding of IgG-coated bacteria activates the neutropil’s phagocitic activity and causes a rapid
surge in intracellular metabolism.
• Complement receptors (CRs) facilitate binding and uptake of immune complexes that are opsonized by active C3 complement protein, namely, C3b. Binding of bacteria or other C3b-coated antigens to CRs triggers phagocytosis, resulting in activation of a neutrophil’s lytic pathways and respiratory burst reactions.
• Scavenger receptors (SRs) are a structurally diverse group of transmembrane glycoproteins that bind to modified (acetylated or oxidized) forms of low-density lipoproteins (LDLs), polyanionic molecules that are often on the surface of both Gram-positive and Gram-negative bacteria and apoptotic bodies. Binding of these receptors increases the phagocitic activity of neutrophils.
• Toll-like receptors, also known as pattern recognition receptors (PRRs), are neutrophil receptors that recognize pathogenic molecules such as endotoxins, lipopolysaccharides, peptidoglycanes, and lipoteichoic acids that are arranged in predictable pathogen-associated molecular patterns (PAMPs) and are commonly expressed on bacterial surfaces and other infectious agents. Like other phagocytic cells, neutrophils possess a variety of tolllike receptors that recognize PAMPs.
Phagocytosed bacteria are killed within phagolysosomes by the toxic reactive oxygen intermediates produced during respiratory burst.
Phagocytosis begins when the neutrophil recognizes and attaches to the antigen. Extended pseudopods of the neutrophil engulf the antigen and internalize it to form a phagosome (see Fig. 10.7). Specific and azurophilic granules fuse with the phagosome membrane, and the lysosomal hydrolases of the azurophilic granules digest the foreign material. During phagocytosis, the neutrophil’s glucose and oxygen utilization increases noticeably and is referred to as the respiratory burst. It results in synthesis of several oxygen- containing compounds called reactive oxygen intermediates (ROIs). They include free radicals such as oxygen and hydroxyl radicals that are used in immobilizing and killing live bacteria within the phagolysosomes. By definition, free radicals possess an unpaired electron within their chemical structure, which makes them highly reactive and therefore capable of damaging intracellular molecules, including lipids, proteins, and nucleic acids. The process by which microorganisms are killed within neutrophils is termed oxygen-dependent intracellular killing. In general,
two biochemical pathways are involved in this process: the first is the phagocyte oxidase (phox) system that utilizes the phagocyte’s nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in the phagolysosome membrane; the second is associated with the lysosomal enzyme myeloperoxidase (MPO) found in the azurophilic granules of neutrophils (Fig 10.. Within the phagocyte oxidase pathway, or phox system, phagocytosis proceeds by signaling the cell to produce sufficient amounts of NADPH needed to generate superoxide anions. Increased glucose uptake and shunting of NADPH metabolism is achieved via the pentose phosphate pathway (also known as pentose shunt). The cytosolic NADPH becomes an electron donor: The NADPH oxidase enzyme complex transports electrons across the membrane to molecular O2 inside the phagolysosome to generate the free radical superoxide anions (O–2). These superoxide anions are converted into ROIs. The superoxide dismutase converts superoxide anions to singlet oxygen (1O2) and hydrogen peroxide (H2O2), which further reacts with superoxide anions to produce bactericidal hydroxyl radicals (OH–) (the neutral form of the hydroxyl ion) and more singlet oxygen molecules (see Fig 10..
Oxygen-dependent killing with MPO involvement occurs when azurophilic granules containing MPO fuse with phagosomes containing phagocytosed bacteria. During the neutrophil’s respiratory burst, MPO, using heme as a cofaccofactor, catalyzes a reaction that produces hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and a chloride anion (Cl–). Hypochlorous acid, which is about 1,000 times more effective in bacterial killing than hydrogen peroxide, is further metabolized to a highly toxic hypochlorite OCl– (bleach) and chlorine (Cl2). Some of the hypochloride may spontaneously break down to yield toxic singlet oxygen (1O2) and chloride ions (Cl–) (see Fig. 10.8 ). In addition, nitric oxide (NO) and other reactive nitrogen intermediates (RNIs) have also been implicated in the intracellular microbial killing mechanisms. NO has been found in neutrophils; however, it is believed that RNI-mediated killing mechanisms do not appear to have a critical role in humans. The main role of neutrophil-derived NO is to induce vasodilatation, which in turn facilitates the migration of neutrophils from blood vessels to surrounding connective tissue.
Phagocytosed bacteria can also be killed by a diverse arsenal of oxygen-independent killing mechanisms utilizing bacteriolytic enzymes and antimicrobial peptides.
In addition to the oxygen-dependent respiratory burst reactions, microorganisms can be killed by bacteriolytic enzymes and cationic antimicrobial peptides that are stored within the granules of the neutrophil’s cytoplasm. These oxygenindependent killing mechanisms are directed toward the bacterial cell membrane, causing its breakdown and leakage.
Neutrophils contain particularly large amounts of cationic antimicrobial proteins such as defensins and antimicrobial peptides called cathelicidins. Similar to lysozymes and
cathepsins stored in the specific granules, these cationic antimicrobial proteins break down the bacterial wall. In addition, lysosomal hydrolytic enzymes that digest bacterial
proteins and lactoferrins that chelate iron from nutritional bacterial pathways contribute to the destruction of the invading bacteria.
These mechanisms are not as efficient as oxygen- dependent killing pathways. Neutrophils from patients with defects in oxygen-dependent pathways, such as those with chronic granulomatous disease (see Folder 10.4), are still able to destroy phagocytosed bacteria to some degree. However, because of the low efficiency of these processes, individuals with these defects are more susceptible to serious infections. After intracellular digestion by the neutrophil, the remnants of degraded material are stored in residual bodies or
exocytosed. Most neutrophils die in this process; the accumulation of dead bacteria and dead neutrophils constitutes the thick exudate called pus. The yellow–green color of the
pus and of mucus secretions (e.g., from infected lungs) comes from the heme pigment of MPO enzyme in azurophilic granules of neutrophils.
Inflammation and wound healing also involve monocytes, lymphocytes, eosinophils, basophils, and fibroblasts
Monocytes also enter the connective tissue as a secondary response to tissue injury. At the site of tissue injury, they transform into macrophages that phagocytose cell and tissue
debris, fibrin, remaining bacteria, and dead neutrophils. Normal wound healing depends on the participation of macrophages in the inflammatory response; they become the
major cell type in the inflammatory site after the neutrophils are spent. At the same time that the macrophages become active at the site of inflammation, fibroblasts near the site and undifferentiated mesenchymal cells in the adventitia of small vessels at the site begin to divide and differentiate into fibroblasts and myofibroblasts that then secrete the fibers and ground substance of the healing wound. Like neutrophils, monocytes are attracted to the inflammatory site by chemotaxis.
Eosinophils are about the same size as neutrophils, and their nuclei are typically bilobed (Fig. 10.9). As in neutrophils, the compact heterochromatin of eosinophils is chiefly adjacent to the nuclear envelope, whereas the euchromatin is located in the center of the nucleus.
Eosinophils are named for the large, eosinophilic, refractile granules in their cytoplasm
The cytoplasm of eosinophils contains two types of granules: numerous, large, elongated specific granules and azurophilic granules (otherwise, the eosinophil contains only a sparse representation of membranous organelles).. Specific granules of eosinophils contain a crystalloid body that is readily seen with the TEM, surrounded by a less electron-dense matrix. These crystalloid bodies are responsible for the refractivity of the granules in the light microscope. They contain four major proteins: an arginine-rich protein called major basic protein (MBP), which accounts for the intense acidophilia of the granule; eosinophil cationic protein (ECP); eosinophil peroxidase (EPO); and eosinophil-derived neurotoxin (EDN). MBP is localized in the crystalloid body; the other three proteins are found in the granule matrix. MBP, ECP, and EPO have a strong cytotoxic effect on protozoans and helminthic parasites; EDN causes nervous system dysfunction in parasitic organisms; histaminase neutralizes the activity of histamine; and arylsulfatase neutralizes leukotrienes secreted by basophils and mast cells.Specific granules also contain histaminase, arylsulfatase, collagenase, and cathepsins.
• Azurophilic granules are lysosomes. They contain a variety of the usual lysosomal acid hydrolases and other hydrolytic enzymes that function in destruction of parasites and hydrolysis of antigen–antibody complexes internalized by the eosinophil.
Eosinophils are associated with allergic reactions, parasitic infections, and chronic inflammation.
Eosinophils develop and mature in the bone marrow. Once released from the bone marrow, they circulate in peripheral blood and then migrate to the connective tissue. Eosinophils are activated by interactions with IgG, IgA, or secretory IgA antibodies.
The release of arylsulfatase and histaminase by eosinophils at sites of allergic reaction moderates the potentially deleterious effects of inflammatory vasoactive mediators. The eosinophil also participates in other immunologic responses and phagocytoses antigen–antibody complexes. Thus, the count of eosinophils in blood samples of individuals with allergies and parasitic infections is usually high. Eosinophils play a major role in host defense against helminthic parasites. They are also found in large numbers in the lamina propria of the intestinal tract and at other sites of potential chronic inflammation (i.e.,lung tissues in patients with asthma).
Basophils are about the same size as neutrophils and are so named because the numerous large granules in their cytoplasm stain with basic dyes.
Basophils are the least numerous of the WBCs, accounting for less than 0.5% of total leukocytes.
Often, several hundred WBCs must be examined in a blood smear before one basophil is found. The lobed basophil nucleus is usually obscured by the granules in stained blood
smears, but its characteristics are evident in electron micrographs (Fig. 10.10). Heterochromatin is chiefly in a peripheral location, and euchromatin is chiefly centrally located;
typical cytoplasmic organelles are sparse. The basophil plasma membrane possesses numerous high-affinity Fc receptors for IgE antibodies. In addition, a specific 39-kilodalton protein called CD40L is expressed on the basophil’s surface. CD40L interacts with a complementary receptor (CD40) on B lymphocytes, which results in increased synthesis of IgE.
The basophil cytoplasm contains two types of granules: specific granules, which are larger than the specific granules of the neutrophil, and nonspecific azurophilic granules.
• Specific granules exhibit a grainy texture and myelin figures when viewed with the TEM. These granules contain a variety of substances, namely, heparin, histamine, heparan sulfate, leukotrienes, IL-4, and IL-13. Heparin, a sulfated glycosaminoglycan, is an anticoagulant. Histamine and heparan sulfate are vasoactive agents that among other actions cause dilation of small blood vessels. Leukotrienes are modified lipids that trigger prolonged constriction of smooth muscles in the pulmonary airways. Interleukin-4 (IL-4) and interleukin-13 (IL-13) promote synthesis of IgE antibodies. The intense basophilia of these specific granules correlates with the high concentration of sulfates within the glycosaminoglycan molecules of heparin and heparan sulfate.
• Azurophilic granules are the lysosomes of basophils and contain a variety of the usual lysosomal acid hydrolases that are similar to those in other leukocytes.
The function of basophils is closely related to that of mast cells.
Basophils are functionally related to, but not identical with, mast cells of the connective tissue. Both mast cells and basophils bind an antibody secreted by plasma cells, IgE, through high-affinity Fc receptors expressed on their cell surface. The subsequent exposure to, and reaction with, the antigen (allergen) specific for IgE triggers the activation of basophils and mast cells and the release of vasoactive agents from cell granules. These substances are responsible for the severe vascular disturbances associated with hypersensitivity reactions and anaphylaxis. Furthermore, both basophils and mast cells are derived from the same basophil–mast cell progenitor (BMCP) cell. If a specific BMCP expresses the granulocyte-related transcription factor CCAAT/enhancer-binding protein (C/EBP), the cell becomes committed to differentiate into a basophil progenitor (BaP) cell. Basophils develop and differentiate in the bone marrow and are released to the peripheral blood as mature cells. In the absence of C/EBP transcription factor, a BMCP cell migrates to the spleen and after further differentiation travels as a mast cell precursor (MPC) to the intestine, where it becomes a mature mast cell.
Lymphocytes are the main functional cells of the lymphatic or immune system
Lymphocytes are the most common agranulocytes and account for about 30% of the total blood leukocytes.
Lymphocytes are essential for immunologic defense of the organism.
In understanding the function of the lymphocytes, one must realize that most lymphocytes found in blood or lymph represent recirculating immunocompetent cells (i.e., cells that have developed the capacity to recognize and respond to antigens and are in transit from one lymphatic tissue to another). In the tissues associated with the immune system, three groups of lymphocytes can be identified according to size: small, medium, and large lymphocytes, ranging in diameter from 6 to 30 μm. The large lymphocytes are either activated lymphocytes, which possess surface receptors that interact with a specific antigen, or natural killer (NK) lymphocytes. In the bloodstream, most lymphocytes are small or medium sized, 6 to 15 μm in diameter. The majority—more than 90%—are small lymphocytes. In blood smears, the mature lymphocyte approximates the size of an erythrocyte. When observed in the light microscope in a blood smear, small lymphocytes have an intensely staining, slightly indented, spherical nucleus. The cytoplasm appears as a very thin, pale blue rim surrounding the nucleus. In general, there are no recognizable cytoplasmic organelles other than an occasional fine azurophilic granule. The TEM reveals that the cytoplasm primarily contains free ribosomes and a few mitochondria. Other organelles are so sparse that they are usually not seen in a thin section. Small, dense lysosomes that correspond to the azurophilic granules seen in the light microscope are occasionally observed; a pair of centrioles and a small Golgi apparatus are located in the cell center, the area of the indentation of the nucleus. In the medium lymphocyte, the cytoplasm is more abundant, the nucleus is larger and less heterochromatic, and the Golgi apparatus is somewhat more developed (Fig. 10.11). Greater numbers of mitochondria and polysomes and small profiles of rough endoplasmic reticulum are also seen in these medium-sized cells. The ribosomes are the basis for the slight basophilia displayed by lymphocytes in stained blood smears.
Three functionally distinct types of lymphocytes are present in the body: T lymphocytes, B lymphocytes, and NK cells.
The characterization of lymphocyte types is based on their function, not on their size or morphology. T lymphocytes (T cells) are so named because they undergo differentiation in the thymus. B lymphocytes (B cells) are so named because they were first recognized as a separate population in the bursa of Fabricius in birds or bursa-equivalent organs (e.g., bone marrow) in mammals. Natural killer (NK) cells develop from the same precursor cell as B and T cells and are so named because they are programmed to kill certain types of transformed cells.
• T cells have a long life span and are involved in cellmediated immunity. T cells are characterized by the presence of cell-surface recognition proteins called T-cell receptors (TCRs), which in a majority of T cells comprise two glycoprotein chains called - and -TCR chains. They express CD2, CD3, CD5, and CD7 marker proteins on their surface; however, they are subclassified on the basis of the presence or absence of CD4 and CD8 proteins. CD4 T lymphocytes possess the CD4 marker and recognize antigens bound to major histocompatability complex II (MHC II) molecules. CD8 T lymphocytes possess the CD8 marker and recognize antigen bound to MHC I molecules.
• B cells have variable life spans and are involved in the production of circulating antibodies. Mature B cells in blood express IgM and IgD and MHC II molecules on their surface. Their specific markers are CD9, CD19,
CD20, and CD24.
• NK cells are programmed during their development to kill certain virus-infected cells and some types of tumor cells. They also secrete an antiviral agent, interferon (IFN-). NK cells are larger than B and T cells (15 μm
in diameter) and have a kidney-shaped nucleus. Because NK cells have several large cytoplasmic granules easily seen by light microscopy, they are also called large granular lymphocytes (LGLs). Their specific markers include CD16, CD56, and CD94. T and B cells are indistinguishable in blood smears and tissue sections; immunocytochemical staining for different types of markers and receptors on their cell surface must be used to identify them. NK lymphocytes can be identified in the light microscope by size, nuclear shape, and presence of cytoplasmic granules; however, immunocytochemical staining for their specific markers is used to confirm microscopic identification.
T and B lymphocytes express different surface molecules.
Although the T and B cells cannot be distinguished on the basis of their morphology, their distinctive surface proteins (CD proteins) can be used to identify the cells with immunolabeling techniques. In addition, immunoglobulins are expressed on the surface of B cells that function as antigen receptors. In contrast, T cells do not have antibodies but express TCRs. These recognition proteins appear during discrete stages in the maturation of the cells within the thymus. In general, the surface molecules mediate or augment specific T-cell functions and are required for the recognition or binding of T cells to antigens displayed on the surface of target cells. In human blood, 60% to 80% of lymphocytes are mature T cells, and 20% to 30% are mature B cells. Approximately 5% to 10% of the cells do not demonstrate the surface markers associated with either T or B cells. These are NK cells and the rare circulating hemopoietic stem cells (see below). The size differences described above may have functional significance; some of the large lymphocytes may be cells that have been stimulated to divide whereas others may be plasma cell precursors that are undergoing differentiation in response to the presence of antigen.
Several different types of T lymphocytes have been identified: cytotoxic, helper, suppressor, and gamma/delta
The activities of cytotoxic, helper, suppressor, and gamma/delta T lymphocytes are mediated by molecules located on their surface. Immunolabeling techniques have made it possible to identify specific types of T cells and study their function.
• Cytotoxic CD8 T cells serve as the primary effector cells in cell-mediated immunity. CD8 cells are specifically sensitized T lymphocytes that recognize antigens through the TCRs on viral or neoplastic host cells. Cytotoxic CD8 T lymphocytes only recognize antigens bound to MHC I molecules. After the TCR binds the antigen–MHC I complex, the cytotoxic CD8T cells secrete lymphokines and perforins that produce ion channels in the membrane of the infected or neoplastic cell, leading to its lysis (see Chapter 14, Lymphatic System). Cytotoxic CD8 T lymphocytes play a significant role in rejection of allografts and in tumor immunology.
• Helper CD4 T cells are critical for induction of an immune response to a foreign antigen. Antigen bound to MHC II molecules is presented by antigen-presenting cells such as macrophages to a helper CD4 T lymphocyte. Binding of the TCR to the antigen–MHC II complex activatesthe helper CD4 T cells. The activated helper CD4 T lymphocytes then produce interleukins (mainly IL-2), which act in an autocrine mode to simulate the proliferation and differentiation of more helper CD4 T lymphocytes. Newly differentiated cells synthesize and secrete lymphokines that affect function as well as differentiation of B cells, T cells, and NK cells. B cells differentiate into plasma cells and synthesize antibody.
• Regulatory (suppressor) T-cells represent a phenotypically diverse population of T lymphocytes that can functionally suppress an immune response to foreign and self-antigen by influencing the activity of other cells in the immune system. The CD4CD25FOXP3 regulator T cells represent a classical example of cells that can downregulate the ability of T lymphocytes to initiate immune responses. The FOXP3 marker indicates an expression of forkhead family transcription factors that are characteristic of many T cells. Furthermore, tumor-associated CD8CD45RO T suppressor cells secrete IL-10 and
also suppress T-cell activation. The suppressor T cells may also function in suppressing B-cell differentiation and in regulating erythroid cell maturation in the bone marrow.
• Gamma/delta ( ) T cells represent a small population of T cells that possess a distinct TCR on their surface. Most T cells have a TCR receptor composed of two glycoprotein chains called - and -
TCR chains. In contrast, T cells possess TCR receptors made up of one -chain and one -chain.
These cells develop in the thymus and migrate into various epithelial tissues (e.g., skin, oral mucosa, intestine, and vagina). Once they colonize an epithelial tissue, they do not recirculate between blood and lymphatic organs. They are also known as intraepithelial lymphocytes. Their location within the skin and mucosa of internal organs allows them to function in the first line of defense against invading organisms.
Monocytes are the precursors of the cells of the mononuclear phagocytotic system.
Monocytes are the largest of the WBCs in a blood smear (average diameter, 18 μm). They travel from the bone marrow to the body tissues, where they differentiate into the various phagocytes of the mononuclear phagocytotic system— that is, connective tissue macrophages, osteoclasts, alveolar macrophages, perisinusoidal macrophages in the liver (Kupffer cells), and macrophages of lymph nodes, spleen, and bone marrow among others. Monocytes remain in the blood for only about 3 days. The nucleus of the monocyte is typically more indented than that of the lymphocyte (Fig. 10.12 ).
The indentation is the site of the cell center where the welldeveloped Golgi apparatus and centrioles are located. Monocytes also contain smooth endoplasmic reticulum, rough endoplasmic reticulum, and small mitochondria. Although these cells are classified as agranular, they contain small, dense, azurophilic granules. These granules contain typical lysosomal enzymes similar to those found in the azurophilic granules of neutrophils.
Monocytes transform into macrophages, which function as antigen-presenting cells in the immune system
During inflammation, the monocyte leaves the blood vessel at the site of inflammation, transforms into a tissue macrophage, and phagocytoses bacteria, other cells, and tissue debris
The monocyte–macrophage is an antigen-presenting cell and plays an important role in immune responses by partially degrading antigens and presenting their fragments on the MHC II molecules located on the macrophage surface of helper CD4 T lymphocytes for recognition.
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