Plasma

Plasma is the fluid component of blood (Figure 6-1). Plasma contains salts and organic compounds (including amino acids, lipids, vitamins, proteins, and hormones). In the absence of anticoagulants, the cellular elements of blood, together with plasma proteins (mostly fibrinogen), form a clot in the test tube. The fluid portion is called serum, which is essentially fibrinogen-free plasma.

Figure 6-1 Blood: Plasma, serum, and cells

Cellular elements of the blood: Red blood cells (erythrocytes)

RBCs, also called erythrocytes (Greek erythros, red; kytos, cell), are non-nucleated, biconcave-shaped cells measuring 7.8 μm in diameter (unfixed). RBCs lack organelles and consist only of a plasma membrane, its underlying cytoskeleton (Figure 6-2), hemoglobin, and glycolytic enzymes.

Figure 6-2 Cell membrane of a red blood cell

RBCs (average number: 4 to 6 × 10⁶ per mm³) circulate for 120 days. Senescent RBCs are removed by phagocytosis or destroyed by hemolysis in the spleen. RBCs are replaced in the circulation by reticulocytes, which complete their hemoglobin synthesis and maturation 1 to 2 days after entering the circulation. Reticulocytes account for 1% to 2% of circulating RBCs. RBCs transport oxygen and carbon dioxide and are confined to the circulatory system.

Clinical significance: Cytoskeletal and hemoglobin abnormalities

Elliptocytosis and spherocytosis are alterations in the shape of RBCs caused by defects in the cytoskeleton. Elliptocytosis, an autosomal dominant disorder characterized by the presence of oval-shaped RBCs, is caused by defective self-association of spectrin subunits, defective binding of spectrin to ankyrin, protein 4.1 defects, and abnormal glycophorin (see Figure 6-2). Spherocytosis is also an autosomal dominant condition involving a deficiency in spectrin. The common clinical features of elliptocytosis and spherocytosis are anemia, jaundice, and splenomegaly (enlargement of the spleen). Splenectomy is usually curative, because the spleen is the primary site responsible for the destruction of elliptocytes and spherocytes.

Hemoglobin genetic defects (α₂βS₂) cause sickle cell anemia and thalassemia (Greek thalassa, sea; observed in populations along the Greek and Italian coasts). Sickle cell anemia results from a point mutation in which glutamic acid is replaced by valine at the sixth position in the β-globin chain. Defective hemoglobin (Hb S) tetramers aggregate and polymerize in deoxygenated RBCs, changing the biconcave disk shape into a rigid and less deformable sickle-shaped cell. Hb S leads to severe chronic hemolytic anemia and obstruction of postcapillary venules (see Spleen in Chapter 10, Immune-Lymphatic System).

Thalassemia syndromes are heritable anemias characterized by defective synthesis of either the α or β chains of the normal hemoglobin tetramer (α₂β₂). The specific thalassemia syndromes are designated by the affected globin chain: α-thalassemia and β-thalassemia. Thalassemia syndromes are defined by anemia caused by defective synthesis of the hemoglobin molecule and hemolysis.

Clinical significance: Erythroblastosis fetalis

Erythroblastosis fetalis is an antibody-induced hemolytic disease in the newborn that is caused by blood group incompatibility between mother and fetus (Figure 6-3 and Box 6-A). This incompatibility occurs when the fetus inherits RBC antigenic determinants that are foreign to the mother. ABO and Rh blood group antigens are of particular interest.

Figure 6-3 Erythroblastosis fetalis: Hemolytic disease of the newborn

Essentially, the mother becomes sensitized to blood group antigens on red blood cells, which can reach maternal circulation during the last trimester of pregnancy (when the cytotrophoblast is no longer present as a barrier, as we discuss in Chapter 23, Fertilization, Placentation, and Lactation) or during childbirth. Within the Rh system, D antigen is the major cause of Rh incompatibility. The initial exposure to the Rh antigen during the first pregnancy does not cause erythroblastosis fetalis because immunoglobulin M (IgM) is produced and IgMs cannot cross the placenta because of their large size.

Subsequent exposure to D antigen during the second or third pregnancy leads to a strong immunoglobulin G (IgG) response (IgGs can cross the placenta).

Rh-negative mothers are given anti-D globulin soon after the delivery of an Rh-positive baby. Anti-D antibodies mask the antigenic sites on the fetal RBCs that may have leaked into the maternal circulation during childbirth. This prevents long-lasting sensitization to Rh antigens.

Granulocytes

These phagocytic cells have a multilobed nucleus and measure 12 to 15 μm in diameter. Their average lifespan varies with cell type. Three types of granulocytes can be distinguished by their cytoplasmic granules:

1. Neutrophils (Figure 6-4). These cells have a lobulated nucleus. Their cytoplasm contains both secondary (specific) and primary granules (see Box 6-C). In stained smears, neutrophils appear very pale pink. Neutrophils, which constitute 60% to 70% of circulating leukocytes, have a lifespan of 6 to 7 hours and may live for up to 4 days in the connective tissue. After leaving the circulation through postcapillary venules, neutrophils act to eliminate opsonized bacteria or limit the extent of an inflammatory reaction in the connective tissue. The mechanism of bacterial opsonization is discussed in Chapter 10, Immune-Lymphatic System.

Figure 6-4 Neutrophil

Enzymes contained in the primary granules (elastase and myeloperoxidase) and secondary granules (lysozyme and other proteases), specific receptors for C5a (produced by the complement system pathway, see Chapter 10, Immune-Lymphatic System), and L-selectin, and integrins (with binding affinity to endothelial cell ligands such as intercellular-adhesion molecules 1 and 2 [ICAM-1 and ICAM-2]) enable the antibacterial and homing function of neutrophils (see Figure 6-9).

Figure 6-5 Eosinophil

Figure 6-6 Basophil

Agranulocytes

Agranulocytes include lymphocytes and monocytes. Agranulocytes have a round or indented nucleus. They contain only lysosomal-type, primary granules.

Lymphocytes are either large (3% of lymphocytes; 9 to 12 μm) or small (97% of lymphocytes; 6 to 8 μm (Figure 6-7) cells. In either case, the nucleus is round and may be slightly indented. The cytoplasm is basophilic, often appearing as a thin rim around the nucleus (see Figure 6-7). A few primary granules may be present. Lymphocytes may live for a few days or several years.

Figure 6-7 Lymphocyte

Lymphocytes are divided into two categories: B lymphocytes (also called B cells) are produced and mature in bone marrow. Antigen-stimulated B cells differentiate into antibody-secreting plasma cells. T lymphocytes (also called T cells) are produced in bone marrow but complete their maturation in the thymus. Activated T cells participate in cell-mediated immunity (for additional details, see Chapter 10, Immune-Lymphatic System).

Monocytes (Figure 6-8) can measure 12 to 20 μm in diameter. Their nucleus is kidney shaped or oval. Cytoplasmic granules are small and may not be resolved on light microscopy. Monocytes circulate in blood for 12 to 100 hours and then enter the connective tissue. In the connective tissue, monocytes differentiate into macrophages, which are involved in bacterial phagocytosis, antigen presentation, and clean-up of dead cell debris. In bone, monocytes differentiate into osteoclasts under the control of osteoblasts (see Chapter 4, Connective Tissue).

Figure 6-8 Monocyte

Figure 6-9 Homing and inflammation

Clinical significance: Homing and inflammation

We have studied in Chapter 1, Epithelium (see Figure 1-13), the molecular principles of homing. We will expand the concept of homing by studying the mechanism of migration of phagocytic neutrophils to the site of infection and inflammation (Figure 6-9).

The first step is the binding of carbohydrate ligands on the surface of the neutrophil to an endothelial selectin (E selectin). This binding determines rolling adhesion of the neutrophil.

The second step is a stronger interaction of neutrophil integrins LFA-1 (lymphocyte function–associated antigen 1) to ICAM-1 on the endothelial cell surface. ICAM-1 is induced by cytokine tumor necrosis factor–α, and interleukin-1 (IL-1) is produced by activated macrophages present at the site of inflammation.

These molecular interactions determine (1) tight binding of the neutrophil, required for stopping rolling; (2) preparing the cell for squeezing between adjacent endothelial cells toward the chemoattractant interleukin-8, produced by inflammatory cells; and (3) transendothelial migration, or diapedesis, facilitated by disrupting the interaction of endothelial cell adhesion molecules such as junctional adhesion molecules (JAMs), endothelial cell cadherin (EC-cadherin), and CD99. The up-regulation of α6β1 integrin by endothelial cell CD99 facilitates penetration of the vascular basal lamina.

Clinical significance of homing: Leukocyte adhesion deficiencies

Cell adhesion proteins play a significant role in immune surveillance, wound healing, tumor metastasis, and tissue morphogenesis. One of the main events in allergic inflammation is the recruitment of inflammatory cells into tissue sites where allergic reactions occur.

Two leukocyte adhesion deficiencies have been described, both characterized by a defect in wound healing, recurrent infections, and marked leukocytosis (increase in the number of leukocytes in blood).

Leukocyte adhesion deficiency I is caused by a defect in the β subunit of the integrin molecule. As a consequence, leukocytes are unable to leave blood vessels to enter the tissue by transendothelial migration. In these patients, inflammatory cell infiltrates are devoid of neutrophils.

In leukocyte adhesion deficiency II, the fucosyl-containing ligands for selectins are absent due to a congenital defect of endogenous fucose metabolism. As shown in Figure 6-9, selectin-carbohydrate interactions have a role in the rolling of leukocytes on an endothelial cell surface, a step required for the transendothelial migration of leukocytes into extravascular areas of inflammation.

Clinical significance: Mast cell-eosinophil interaction in asthma

We have already seen that both mast cells and eosinophils are immigrant cells of the connective tissue. These two cell types have a significant role in the pathogenesis of asthma.

Asthma, a condition in which extrinsic (allergens) or intrinsic (unknown) factors trigger variable air obstruction of the respiratory bronchi and bronchioles, provides a good example of mast cell–eosinophil interaction.

When mast cells degranulate and release chemical mediators, eosinophils and neutrophils are attracted from blood vessels into the connective tissue of the respiratory mucosa. Eosinophils, in turn, release additional mediators (leukotriene B₄ and others) to enhance bronchoconstriction and edema. The release of eosinophil cationic protein and major basic protein into the bronchial lumen damages the epithelial cell lining and disturbs mucociliary function (Figure 6-10).

Figure 6-10 Mast cell-eosinophil interaction in asthma

Clinical significance: Thrombocytopenia

About 300,000 platelets per microliter of blood circulate for 8 to 10 days. Platelets promote blood clotting and help to prevent blood loss from damaged vessels. A reduction in the number of platelets in blood (thrombocytopenia) leads to increased susceptibility to bleeding. Thrombocytopenia is defined by a decrease in the number of platelets to less than 150,000/μL of blood. Spontaneous bleeding is observed with a platelet count of 20,000/μL. Thrombocytosis defines an increase in the number of platelets circulating in blood.

Thrombocytopenia can be caused by a decrease in the production of platelets, an increase in the destruction of platelets (determined by antibodies against platelets or megakaryocyte antigens [autoimmune thrombocytopenic purpura, ITP] or drugs—for example, penicillin, sulfonamides, and digoxin), and aggregation of platelets in the microvasculature (thrombotic thrombocytopenic purpura [TTP]), probably a result of pathologic changes in endothelial cells producing procoagulant substances.

Deficiency of the glycoprotein 1b—factor IX complex, or von Willebrand’s factor, a protein associated with factor VIII, leads to two congenital bleeding disorders, Bernard-Soulier syndrome and von Willebrand’s disease, respectively (see Figures 6-11 to 6-13) (see Box 6-D). These two diseases are characterized by the inability of platelets to attach to vascular subendothelial surfaces. The glycoprotein 1b—factor IX—von Willebrand’s factor complex is relevant for the aggregation of normal platelets when they are exposed to injured subendothelial tissues.

Figure 6-12 Blood clotting or hemostasis

Figure 6-13 Phases of blood clotting

Gray platelet syndrome, an autosomal dominant disease characterized by macrothrombocytopenia (thrombocytopenia with increased platelet volume), is due to a reduction in the content of alpha granules.

MYH9 (myosin heavy chain 9)-related disorders are also associated with macrothrombocytopenia. A defect in the MYH9 gene, which encodes nonmuscle myosin heavy chain IIA, an isoform expressed in platelets and neutrophils, determines defective production of platelets during the formation of proplatelets.

Clinical significance: Hemostasis and the blood clotting cascade

The blood clotting or coagulation cascade depends on the sequential activation of proenzymes to enzymes and the participation of endothelial cells and platelets to achieve hemostasis or arrest of bleeding. Hemostasis occurs when fibrin is formed to reinforce the platelet plug (Figure 6-12).

The blood clotting cascade has the following characteristics:

The extrinsic pathway is triggered by damage outside a blood vessel and is set in motion by the release of tissue factor. The intrinsic pathway is stimulated by damage to components of the blood and blood vessel wall. It is induced by contact of factor XII to subendothelial collagen. This contact results from damage to the wall of a blood vessel.

Extrinsic and intrinsic pathways converge to a crucial step in which fibrinogen is converted to fibrin, which forms mesh that enables platelets to attach. The convergence starts with the activation of factor X to factor Xa, together with activated factor Va, resulting in the cleavage of prothrombin to thrombin. The initial hemostatic plug consists of a platelet scaffold for the conversion of prothrombin to thrombin, which changes fibrinogen into fibrin (see Figure 6-12).

Fibrinogen, produced by hepatocytes, consists of three polypeptide chains, which contain numerous negatively charged amino acids in the amino terminal. These characteristics allow fibrinogen to remain soluble in plasma. After cleavage, the newly formed fibrin molecules aggregate forming a mesh. Fibrin, with the addition of plasma fibronectin, stabilizes the blood clot (Figure 6-13).

Hematopoietic cell populations

The bone marrow consists of three major populations (see Figure 6-16): (1) the hematopoietic stem cells, capable of self-renewal; (2) committed precursor cells, responsible for the generation of distinct cell lineages; and (3) maturing cells, resulting from the differentiation of the committed precursor cell population.

Hematopoietic stem cells can self-renew and produce two committed precursor cells: the myeloid stem cell and the lymphoid stem cell, that develop into distinct cell progenies. Self-renewal is an important property of hematopoietic stem cells. Self-renewal preserves the pool of stem cells and is critical for feeding common myeloid progenitor and common lymphoid progenitor into the differentiation or maturation pathway.

Hematopoietic stem cells are difficult to identify, mainly because they represent approximately 0.05% of total hematopoietic cells (about 10⁶ to 10⁷ stem cells). In bone marrow transplantation, only 5% of the normal hematopoietic stem cells are needed to repopulate the entire bone marrow. Hematopoietic stem cells cannot be identified by morphology, but they can be recognized by specific cell surface markers (c-kit and Thy-1). CD34⁺ committed precursor cell populations, also containing CD34⁻ hematopoietic stem cells, are generally used for transplantation in the clinical treatment of malignant diseases with chemotherapeutic agents that deplete a certain group of committed precursor cells.

Myeloid and lymphoid stem cells are multipotential cells (see Figure 6-16). They are committed to the formation of cells of the blood and lymphoid organs.

Five colony-forming units (CFUs) derive from the myeloid stem cell: the erythroid CFU, the megakaryocyte CFU, the basophil CFU, the eosinophil CFU, and the granulocyte-macrophage CFU. The erythroid CFU produces red blood cells. The megakaryocyte CFU generates platelets. The granulocyte-macrophage CFU produces both monocytes and neutrophils. Basophils and eosinophils derive from the basophil and eosinophil CFUs, respectively. The lymphoid stem cell gives rise to T cell and B cell precursors.

Clinical significance: Hematopoietic growth factors

Hematopoietic growth factors control the proliferative and maturational phases of hematopoiesis. In addition, they can extend the life span and function of a number of cells produced in the bone marrow. Several recombinant forms are available for clinical treatment of blood disorders.

Hematopoietic growth factors, also known as hematopoietic cytokines, are glycoproteins produced in the bone marrow by endothelial cells, stromal cells, fibroblasts, developing lymphocytes, and macrophages. Hematopoietic growth factors are also produced outside the bone marrow.

There are three major groups of hematopoietic growth factors: (1) colony-stimulating factors, (2) erythropoietin (Figure 6-17) and thrombopoietin (Greek thrombos, clot; poietin, to make), and (3) cytokines (primarily interleukins).

Figure 6-17 Erythropoietin

Colony-stimulating factors are so named because they are able to stimulate committed precursor cells to grow in vitro into cell clusters or colonies. Interleukins are produced by leukocytes (mainly lymphocytes) and affect other leukocytes (paracrine mechanism) or themselves (autocrine mechanism).

Hematopoietic cells express distinct patterns of growth factor receptors as they differentiate. Binding of the ligand to the receptor leads to a conformational change, activation of intracellular kinases, and the final induction of cell proliferation (see Chapter 3, Cell Signaling).

We discuss the roles of specific hematopoietic growth factors when we analyze each cell lineage.

Erythroid lineage

Erythropoiesis includes the following sequence (Figure 6-18): proerythroblast, basophilic erythroblast, polychromatophilic erythroblast, orthochromatic erythroblast, reticulocyte, and erythrocyte.

Figure 6-18 Erythroid lineage

The major regulator of erythropoiesis is erythropoietin (EPO) (see Figure 6-17), a glycoprotein produced primarily (90%) in the kidneys (juxtatubular interstitial cells in the renal cortex) in response to hypoxia (a decrease in oxygen level in inspired air or tissues).

Renal juxtatubular interstitial cells sense oxygen levels through oxygen-dependent prolyl hydroxylase, a protein that hydroxylates the transcription factor hypoxia-inducible factor 1α (HIF-1α) to repress the activity of the erythropoietin gene. Under conditions of low oxygen tension, the hydroxylase is inactive and nonhydroxylated HIF-1α can drive the production of erythropoietin.

Erythropoietin stimulates the proliferation of erythroid progenitor cells by decreasing the levels of cell cycle inhibitors and increasing cyclins and the antiapoptotic protein Bclx_L. Erythropoietin is also produced by neurons and glial cells in the central nervous system and in the retina. The administration of erythropoietin exerts a protective effect on neurons after ischemia (stroke).

Erythropoietin production in chronic renal diseases is severely impaired. Recombinant erythropoietin can be administered intravenously or subcutaneously for the treatment of anemia caused by a decrease in the production of erythropoietin by the kidneys. The effectiveness of erythropoietin treatment can be monitored by an increase of reticulocytes in circulating blood. Reticulocytes can be identified by the supravital stain of residual polyribosomes forming a reticular network (Figure 6-19).

Figure 6-19 Erythroid lineage

Polychromatophilic erythroblasts are erythropoietin-independent, mitotically active, and specifically involved in the synthesis of hemoglobin. Derived orthochromatic erythroblasts, reticulocytes, and mature RBCs are postmitotic cells (not involved in mitosis).

Leukopoiesis

Leukopoiesis (Greek leukos, white; poietin, to make) results in the formation of cells belonging to the granulocyte and agranulocyte series. The granulocyte lineage (Figure 6-20) includes the myeloblast, promyelocyte, myelocyte, metamyelocyte, band cell, and mature form. The granulocyte-macrophage precursor gives rise to neutrophils and monocytes. The myeloid stem cell generates eosinophil and basophil progenies. Agranulocytes include lymphocytes and monocytes.

Figure 6-20 Myeloid lineage

Granulocytes

Neutrophilic and macrophage cell lines share a common precursor cell: the granulocyte-macrophage CFU (see Figure 6-20). Eosinophils and basophils derive from independent eosinophil and basophil CFUs. Neutrophil, eosinophil, and basophil granulocytes follow a similar pattern of proliferation, differentiation, maturation, and storage in the bone marrow. Details of these processes are better recognized for neutrophils, the most abundant granulocyte in the bone marrow and blood. It takes 10 to 14 days for neutrophils to develop from early precursors, but this timing is accelerated in the presence of infections or by treatment with granulocyte colony-stimulating factor (CSF) or granulocyte-macrophage CSF (see below).

Myeloblasts, promyelocytes, and myelocytes are mitotically dividing cells; metamyelocytes and band cells cannot divide but continue to differentiate (see Figure 6-20). A typical feature of the maturation process of granulocytes is the appearance of primary (azurophilic) granules and “specific” or secondary granules in the cytoplasm (Figures 6-21 and 6-22).

Figure 6-21 Myeloid lineage

Figure 6-22 Myeloid lineage: Cell types

Myeloblasts are undifferentiated cells lacking cytoplasmic granules. Promyelocytes and myelocytes display primary granules in cells of the neutrophil, eosinophil, and basophil series. Secondary granules appear in myelocytes. Primary granules do not transform into specific granules. Primary granules persist as such throughout the cell differentiation sequence (see Figure 6-22).

Eosinophils exhibit the same maturation sequence as neutrophils. Eosinophil-specific granules are larger than neutrophil granules and appear refractile under the light microscope. Eosinophilic granules contain eosinophil peroxidase (with antibacterial activity) and several cationic proteins (major basic protein, and eosinophil cationic protein, with antiparasitic activity).

Basophils are distinguished by their large, coarse, and darkly stained granules that fill the cytoplasm and often obscure the nucleus (Figure 6-23). The granules contain peroxidase, heparin, and histamine as well as kallikrein, a substance that attracts eosinophils.

Figure 6-23 Myeloid lineage: Basophil

We discuss in Chapter 4, Connective Tissue, that mast cells are structurally similar to basophils. However, mast cells are larger cells and are found in tissues, close to blood vessels. A notable difference is that mast cells contain serotonin and 5-hydroxytryptamine, which basophils do not contain. In addition, mast cells discharge their granules into the extracellular space in contrast with basophils, which usually undergo diffuse internal degranulation.

Agranulocytes: Lymphocytes

Lymphocytes constitute a heterogeneous population of cells that differ from each other in terms of origin, life span, preferred sites of localization within lymphoid organs, cell surface markers, and function.

The pluripotent stem cell gives rise to all hematopoietic cells, including lymphocytes phocytes of the B and T cell lineage. B cells mature in the bone marrow and then migrate to other lymphoid organs. T cells complete their maturation in the thymus and then migrate to specific lymphoid organs.

A lymphoblast gives rise to a prolymphocyte, an intermediate stage that precedes the mature lymphocyte. B and T lymphocytes are nonphagocytic cells. They are morphologically similar but functionally different, as discussed in Chapter 10, Immune-Lymphatic System.

Lymphoblasts (8 to 12 μm in diameter) are the precursors of the lymphocytes. A lymphoblast has an uncondensed nucleus with a large nucleolus. The cytoplasm contains many polyribosomes and a few cisternae of the endoplasmic reticulum.

Lymphocytes (10 μm in diameter or less) contain a round or slightly indented condensed nucleus. The nucleolus is not visible. The cytoplasm is moderately basophilic and devoid of granules.

Monocytes

Monocytes derive from the granulocyte-macrophage CFU. We have already discussed that the granulocyte-macrophage CFU gives rise to the neutrophil lineage and the macrophage lineage. Under the influence of a specific CSF, each precursor cell establishes its own hierarchy: the granulocyte colony-stimulating factor (G-CSF) takes the granulocyte precursor cell into the myeloblast pathway; the granulocyte-macrophage colony-stimulating factor (GM-CSF) guides the monocyte precursor cell into the monoblast pathway, leading to the production of peripheral blood monocytes and tissue macrophages. Receptors for the macrophage-stimulating factor (M-CSF) are restricted to the monocyte lineage (see Osteoclastogenesis in Chapter 5, Osteogenesis).

Monoblasts (14 μm in diameter) are morphologically similar to myeloblasts. The monoblast is present in the bone marrow and is difficult to identify with certainty. The cytoplasm is basophilic and the nucleus is large and displays one or more nucleoli. The following cell in the series is the promonocyte.

Promonocytes (11 to 13 μm in diameter) contain a large nucleus with a slight indentation and uncondensed chromatin. A nucleolus may be visualized. The basophilic cytoplasm, due to polyribosomes, contains primary granules (lysosomes with peroxidase, arylsulfatase, and acid phosphatase). The primary granules are smaller and fewer than in promyelocytes. Both monoblasts and promonocytes are mitotically active cells.

Monocytes (12 to 20 μm in diameter) in the bone marrow and the blood have a large indented nucleus found in the central portion of the cytoplasm (Figure 6-24). Granules (primary lysosomes) and small vacuoles are typical features. Lysosomes lack peroxidase but contain other proteases and hydrolases. Monocytes are motile in response to chemotactic signals and attach to a surface.

Figure 6-24 Origin and fate of monocytes

Macrophages (15 to 80 μm in diameter) constitute a population of emigrated blood monocytes that differentiate in tissues (lungs, spleen, liver, lymph node, peritoneum, gastrointestinal tract, and bone [osteoclasts]) in response to local conditions.

The structural and functional characteristics of tissue macrophages are discussed in Chapter 4, Connective Tissue. In Chapter 11, Integumentary System, we discuss the antigenic reactivity of monocyte-derived Langerhans cells in epidermis. In Chapter 17, Digestive Glands, we explore the important role of Kupffer cells in liver function, and in Chapter 10, Immune-Lymphatic System, we examine the phagocytic properties of macrophages in spleen.

Clinical significance: Colony-stimulating factors and interleukins

G-CSF is a glycoprotein produced by endothelial cells, fibroblasts, and macrophages in different parts of the body. The synthetic form of G-CSF (known as filgrastim or lenograstim) causes a dose-dependent increase of neutrophils in the blood. G-CSF is used for the treatment of neutropenia (neutrophil + Greek penia, poverty; small numbers of neutrophils in circulating blood) after cancer chemotherapy, after bone marrow transplantation, to facilitate an increase of neutrophils, and in the treatment of chronic neutropenia.

GM-CSF is also a glycoprotein produced by endothelial cells, T cells, fibroblasts, and monocytes that stimulates the formation of neutrophils, eosinophils, basophils, monocytes, and dendritic cells (Figure 6-25). However, GM-CSF is less potent than G-CSF in increasing the levels of neutrophils during neutropenia. As is the case with G-CSF, a synthetic form of GM-CSF (sargramostim or molgramostim) is available for the treatment of neutropenia.

Figure 6-25 Hematopoietic growth factors regulating the myeloid lineage

Interleukins have a relevant function in the formation and function of type B and T cells as we discuss in Chapter 10, Immune-Lymphatic System. IL-3 stimulates proliferation of hematopoietic stem cells and acts together with other growth factors, including stem cell factor, thrombopoietin, IL-1, IL-6, and Flt3 (fms-like tyrosine kinase 3) ligand (see Figure 6-25)• IL-5 acts specifically on the eosinophil progeny.

Platelets and megakaryocytes

The precursor cell of the platelet (also called thrombocyte; Greek thrombos, clot) is the megakaryoblast, a cell derived from the megakaryocyte CFU (see Figure 6-16).

The megakaryoblast (15 to 50 μm in diameter) displays a single kidney-shaped nucleus with several nucleoli. The megakaryoblast enlarges to give rise to the promegakaryocyte (20 to 80 μm in diameter) with an irregularly shaped nucleus and a cytoplasm rich in azurophilic granules. The promegakaryocyte forms the mature megakaryocyte.

The megakaryocyte (35 to 160 μm in diameter; Figure 6-26) contains an irregularly lobed nucleus produced by an endomitotic nuclear division process in which nuclear divisions occur without cell division (polyploid nucleus). Nucleoli are not detected.

Figure 6-26 Megakaryocyte and the origin of platelets

The megakaryocyte can be mistaken for the osteoclast, another large cell in bone that is multinucleated instead of multilobed. The cytoplasm shows a network of demarcation zones formed by the invagination of the plasma membrane of the megakaryocyte. The coalescence of the demarcation membranes results in the formation of the plasma membrane of proplatelets, which fragment into platelets.

Platelets play important roles in maintaining the integrity of blood vessels (see Figure 6-12). Keep in mind that platelet activation during hemostasis involves sequentially:

Clinical significance: Thrombopoietin

Thrombopoietin is produced in the liver, has a similar structure to erythropoietin, and stimulates the development of megakaryocytes from the megakaryocyte CFU into platelets. Deficiencies in thrombopoietin cause thrombocytopenia. An excess of thrombopoietin causes thrombocytosis.

Platelets bind and degrade thrombopoietin, a process that autoregulates platelet production.

Clinical significance: Stem cell factor (also known as c-kit ligand)

Stem cell factor (SCF) is a ligand protein produced by fetal tissues and stromal cells of the bone marrow that binds to the stem cell factor receptor (c-kit receptor), a tyrosine kinase.

Stem cell factor exists in two forms: membrane-associated and soluble forms, the latter generated by proteolytic cleavage of the membrane-associated protein. The c-kit receptor has an extracellular domain consisting of five immunoglobulin motif repeats responsible for stem cell factor binding and dimerization (Figure 6-27). Binding of stem cell factor induces the dimerization of the c-kit receptor, followed by autophosphorylation. Autophosphorylated c-kit receptor is the docking site of specific signaling molecules.

Figure 6-27 c-kit receptor

The intracellular domain has an adenosine triphosphate (ATP) binding site and a catalytic site. The tyrosine kinase inhibitor imatinib binds to the ATP binding site and prevents the phosphorylation of substrates involved in the activation of downstream signaling. Imatinib shows remarkable results in the treatment of chronic myeloid leukemia.

The stem cell factor ligand by itself is a weak stimulator of hematopoiesis but makes hematopoietic stem cells responsive to other cytokines (see Figure 6-25). It does not induce the formation of cell colonies by itself. Flt3 (fms-like tyrosine kinase 3) ligand is closely related to c-kit receptor and stem cell factor. Similar to stem cell factor, Flt3 ligand acts on the pluripotent stem cell in synergy with thrombopoietin, stem cell factor, and interleukins.

The stem cell factor receptor is expressed by the c-kit proto-oncogene. A mutation in genes expressing the components of the stem cell factor receptor–ligand complex causes anemia and affects the development of melanocytes in skin and the survival and proliferation of primordial germinal cells in the developing ovaries and testes (see Chapter 21, Sperm Transport and Maturation). Stem cell factor is potentially useful for the treatment of inherited and acquired disorders of hematopoiesis as well as in bone marrow transplantation.

In Chapter 4, Connective Tissue, we note that mast cells derive from a bone marrow precursor. The storage and release of histamine- and heparin-containing granules from mast cells are affected in mutants lacking stem cell factor.

Clinical significance: Transferrin and iron metabolites

In addition to erythropoietin, the formation of RBCs is highly dependent on iron metabolism and the water-soluble vitamins folic acid (folacin) and vitamin B₁₂ (cobalamin).

Iron is involved in the transport of oxygen and carbon dioxide. Several iron-binding proteins store and transport iron, for example, hemoglobin in RBCs, and myoglobin in muscle tissue. Iron is coupled to heme (a molecule synthesized in the bone marrow, with one ferrous ion, Fe²⁺, bound to a tetrapyrrolic ring) and hematin (with one ferric ion, Fe³⁺, bound to a protein).

Transferrin, a serum protein produced in the liver, and lactoferrin, a protein present in maternal milk, are nonheme proteins involved in the transport of iron (Figure 6-28). Transferrin complexed to two Fe³⁺ ions is called ferrotransferrin. Transferrin devoid of iron is known as apotransferrin.

Figure 6-28 Uptake of iron by internalization of transferrin

The iron-containing transferrin binds to a specific cell surface receptor that mediates the internalization of the transferrin ligand–transferrin receptor complex. The transferrin receptor is a transmembrane dimer with each sub unit binding to a transferrin molecule. The internalization of the transferrin-receptor complex is dependent on receptor phosphorylation triggered by Ca²⁺-calmodulin and the protein kinase C complex.

Inside the cell, iron is released within the acidic endosomal compartment and the receptor-apotransferrin (iron-free) complex returns to the cell surface where apotransferrin is released to be reutilized in blood plasma.

Ferritin, a major protein synthesized in the liver, is involved in the storage of iron. A single ferritin molecule has the capacity to store up to 4500 iron ions. When the storage capacity of ferritin is exceeded, iron is deposited as hemosiderin. Ferritin with little iron is called apoferritin.

Patients with the heritable disorder idiopathic hemochromatosis, characterized by excessive iron absorption and tissue deposits, require periodic withdrawals of blood and the administration of iron chelators to facilitate the excretion of complexed iron in the urine. A decrease in iron by excessive menstrual flow or gastrointestinal bleeding determines a reduction in hemoglobin-containing iron. RBCs are smaller (microcytic anemia) and underpigmented (hypochromic anemia).

Folic acid regulates the folate metabolism leading to the increased availability of purines and deoxythymidine monophosphate (dTMP) required for DNA synthesis.

Vitamin B₁₂ (known as extrinsic factor) binds to intrinsic factor, a protein produced by the parietal cells in the gastric glands. The vitamin B₁₂ –intrinsic factor complex binds to specific receptor sites in the ileum, transported across enterocytes, and released in blood, where it binds to the transport protein trans-cobalaphilin III.

A decrease in vitamin B₁₂, due mainly to insufficient production of intrinsic factor or hydrochloric acid in the stomach, or both, can affect folate metabolism and folate uptake, thereby impairing DNA synthesis in bone marrow.

Vitamin B₁₂ deficiency is rare because the liver stores up to a 6-year supply of vitamin B₁₂. Under deficiency conditions, the maturation of the erythroid cell progeny slows down, causing abnormally large RBCs (megaloblasts) with fragile cell membranes, resulting in the destruction of RBCs (megaloblastic anemia; see Box 6-E).

Concept mapping

Blood and Hematopoiesis