Bernard Feldman United States
In the mammalian fetus, hematopoiesis takes place initially in the yolk sac and later in the liver and the spleen.(1) Islands of hematopoiesis develop in these tissues and then involute as the marrow becomes the primary site for blood cell forma�tion by the seventh month of fetal development.(2) Barring serious damage such as that which occurs with myelofibro�sis or radiation injury, the bone marrow remains the site for blood cell formation throughout the rest of life. During mammalian growth there is active hematopoiesis in the marrow spaces of the central axial skeleton (i.e., ribs, vertebrae, and pelvis) and the extremities extending to the carpus, tarsus, and the calvarium. With normal growth and development, hematopoiesis gradually withdraws from the periphery. This change is reversible, however; distal marrow extension can occur with intensive stimulation, as occurs with severe hemolytic anemias, long-term administration of hematopoietic growth factors, and hematologic malignan�cies. The term medullary hematopoiesis refers to the pro�duction of blood cells in the bone marrow; extramedullary hematopoiesis indicates blood cell production outside the marrow in the spleen, liver, and other locations such as the lymphoid tissue (especially in cats).
ORGANIZATION OF HEMATOPOIETIC TISSUES
The medullary space in which hematopoietic cells develop contains, normally, many adipocytes and a rich vascular supply. Vascular endothelial cells, marrow fibroblasts, and stromal cells are important sources for the matrix proteins that provide structure to the marrow space and for production of the hematopoietic growth factors that stimulate cell proliferation.(3) The vascular endothelial cells also form an important barrier that keeps immature cells in the marrow and permits mature hematopoietic elements to enter the blood. The adipocytes may influence hematopoiesis through their effects on the metabolism of androgens and estrogens. Marrow macrophages serve to remove effete or apoptotic cells, as well as to clear the blood of foreign materials when it enters the marrow. Osteoblasts and osteoclasts maintain and remodel the surrounding can�cellous bone and the calcified lattice, which crisscrosses the marrow space.(3)
The thymus, lymph nodes, mucosa-associated lymphatic tissues (MALT), and the spleen have multiple hematopoietic functions. Early in development, they are major sites for hematopoiesis. In adulthood, they are principally sites for lymphocyte development, processing of antigens, develop�ment of effector T cells, and antibody production. In the myeloproliferative disorders, the size and cellular architecture of these tissues are de�ranged, leading to many of the clinical manifestations of these disorders.
Hematopoietic Stem Cells. All cells of the hematopoietic system are derived from common precursor cells, the hematopoietic stem cells.(4) These cells are difficult to identify, in part because they normally represent only about 0.05 percent of marrow cells.(5) Through self-renewal, this population is maintained without depletion despite giving rise to approximately 1010 erythrocytes and 109 leukocytes an hour for the lifetime of an individual.(6) Utilizing monoclonal antibodies that recognize specific cell surface molecules expressed selectively on developing hematopoietic cells and other specialized techniques, the stem cells can now be separated from other marrow cells. With these methods, the human hematopoietic stem cell has been found to be positive for CD34, c-kit, and thy-1 and negative for HLA-DR, CD15, and CD77.(6) For clinical purposes, CD34+ progenitor cell populations,(7) which contain stem cells and some more mature cells, are often used for hematopoiet�ic stem cell transplantation.
Stem cells give rise to daughter cells, which undergo irreversible commitment to differentiation along various hematopoietic cell lineages. Many aspects of the earliest steps in this differentiation process are not well understood. With lineage commitment, however, differentiation, maturation, and release of cells to the blood come under the control of well-defined hematopoietic growth factors. These growth factors have overlapping activities for the early phases of differentiation.(8) Later in develop�ment, some growth factors are lineage specific, meaning that they govern the maturation and deployment of single lineages. Erythropoietin (EPO), thrombopoietin (TPO), and granulocyte colony-stimulating factor (G-CSF) are the best-characterized lineage-specific factors.
Hematopoietic Growth Factors. The hematopoietic growth factors, also referred to as hematopoietic cytokines, are a family of glycoproteins pro�duced in the bone marrow by endothelial cells, stromal cells, fibroblasts, macrophages, and lymphocytes and also produced at distant sites from which they are transported to the marrow through the blood.(9) The naming of these factors is somewhat confusing. Erythropoietin and thrombopoietin derive part of their names from the Greek word poiesis, meaning to make. The colony-stimulating factors were first recognized because of their capacity to stimulate early hematopoietic cells to grow into clusters and large colonies in tissue culture systems.(10) Interleukin is a term used to describe factors that are produced by leukocytes and that affect other leukocytes. This is a large family of factors predominantly governing lymphocytopoiesis, but many members also have broad effects on other lineages. The discovery of new growth factors and the biologic consequences of deficiencies or excesses of these factors continues to evolve rapidly.
Hematopoietic cells have distinctive patterns of expression of growth factor receptors, and the patterns evolve as the cells differentiate.(11) Each growth factor binds only to its specific receptor.(12) It is now known that for some growth factors, there is sharing of components of the receptor (e.g., interleukin-3 [IL-3], IL-5, and granulocyte-macrophage colony-stimulating factor [GM-CSF] share a common ß chain of their receptor), but specificity comes from other unique or private components of the receptor.(13) Binding of the ligand to the receptor leads to a conformational change, activation of intracellular kinases, and, ultimately, the triggering of cell proliferation.(14) For some growth factors, these pathways are well defined; for others, the pathways are still unclear
Hematopoietic growth factors not only stimulate cell proliferation but also prolong cell survival; that is, they have antiapoptotic effects.(15) For some lineages, such as neutrophils and monocytes, there are growth factor receptors on fully mature cells, and exposure of these cells to the factors primes the cells for an enhanced responsiveness to bacteria or other stimulators of their metabolic activity. Thus, for cells of the neutrophil lineage, the growth factors G-CSF and GM-CSF can stimulate early hematopoietic cell proliferation, increase the number of cells produced by the marrow, prolong the life span of these cells, and augment cell functions.(16)
Erythropoietin. The peritubular interstitial cells located in the inner cortex and outer medulla of the kidney are the primary site for erythropoietin production.(17) In response to hypoxia, transcription of the erythropoietin gene in these cells increases, resulting in increased secretion of erythropoietin. The protein is then transported through the blood to the marrow to stimulate erythropoiesis. With renal failure, erythropoietin production is severely impaired. In infections and many chronic inflammatory conditions, the erythropoietin response is blunted and erythropoietin levels are low.
Erythropoietin is a glycosylated protein that modulates erythropoiesis by affecting several steps in red cell development.(18) The most primitive identifiable erythroid cells, the burst-forming unit-erythroid (BFU-E), are relatively insensitive to erythropoietin. More mature cells, the colony-forming unit-erythroid (CFU-E), are very sensitive. Erythropoietin treatment prolongs survival of erythroid precursors, shortens the time between cell divisions, and increases the number of cells produced from individual precursors.(17)
Erythropoietin can be administered intravenously or subcutaneously for the treatment of anemia caused by inadequate endogenous production of erythropoietin.(19) Treatment is maximally effective when the marrow has a generous supply of iron and other nutrients, such as cobalamin and folic acid. The most easily monitored immediate effect of increased endogenous or exogenous erythropoietin is an increase in the blood reticulocyte count. Normally, as red cell precursors mature, the cells extrude their nucleus at the normal blast stage. The resulting reticulocytes, identified by the supravital stain of their residual ribosomes, persist for about three days in the marrow and one day in the blood. Erythropoietin shortens the transit time through the marrow, leading to an increase in the number and proportion of blood reticulocytes within a few days.(17)
In some conditions, particularly chronic inflammatory disease, the effectiveness of erythropoietin can be predicted from measurement of the serum erythropoietin concentration by immunoassay.(20) It may be cost-effective to measure the concentration before initiating treatment in patients with anemia attributable to suppressed erythropoietin production, such as patients with infection, cancer, renal disease and other chronic inflammatory diseases. Species-specific erythropoietin may be essential.(21-24)
Thrombopoietin. The development of megakaryocytes from hematopoietic stem cells and the level of platelets in the blood are governed by thrombopoietin.(25) Thrombopoietin (TPO) is produced primarily by the liver and has structural similarities to erythropoietin.(26) Plasma thrombopoietin levels are inversely related to the blood platelet count.(27) Deficiencies of thrombopoietin cause thrombocytopenia, and excesses cause thrombocytosis. Recombinant human thrombopoietin is under investigation for the treatment of diverse causes of thrombocytopenia; but, as yet, in human medicine is not approved for clinical use.
Granulocyte Colony-Stimulating Factor is a glycosylated protein produced by monocytes, macrophages, fibroblasts, stromal cells, and endothelial cells throughout the body.(28) It stimulates the growth and differentiation of neutrophils both in vitro and in vivo. G-CSF levels are normally very low or undetectable but increase with bacterial infections or after administration of bacterial endotoxin.(16) G-CSF (the synthesized form is known as filgrastim or lenograstim) administration causes a dose-dependent increase in the blood neutrophil counts in normal persons(29); animals deficient in G-CSF have neutropenia.(30) Like erythropoietin, G-CSF administration accelerates development of neutrophils in the bone marrow, shifting them at an earlier stage than normal from the marrow to the blood.
In human medicine G-CSF is approved for treatment of neutropenia after cancer chemotherapy, for acceleration of neutrophil recovery after bone marrow transplantation, for mobilization of hematopoietic progenitor cells from the marrow to the blood for hematopoietic transplantation, and for treatment of severe chronic neutropenia.(28) It is widely used to treat other neutropenic conditions. Side effects are principally musculoskeletal pain and headaches during the period of rapid marrow expansion soon after therapy is initiated. Other side effects are uncommon. In veterinary medicine both species specific G-CSF and rHG-CSF have been used, as above, and in the treatment of parvoviral enteritis and bovine mastitis.
Granulocyte-Macrophage Colony-Stimulating Factor is a glycosylated protein produced by many types of cells, including T cells.(13) GM-CSF stimulates formation of neutrophils, monocytes, and eosinophils and may also enhance the growth of early cells of other lineages.(10) In contrast to G-CSF, GM-CSF concentrations generally do not increase with infections or acute inflammatory conditions,(31) and neutropenia does not result from deficiencies of GM-CSF.(32) The marrow effects of G-CSF and GM-CSF are similar, but GM-CSF is less potent in elevating the blood neutrophil count. GM-CSF (the synthesized form is known as sargramostim or molgramostim) is approved in human medicine in the United States for acceleration of marrow recovery after bone marrow transplantation and has been widely used to accelerate recovery after chemotherapy and for mobilization of progenitor cells from the marrow. Its side effects include bone and musculoskeletal pain and injection-site reactions.
Other Growth Factors. Several other hematopoietic growth factors are under development. IL-3 acts at an early phase in hematopoiesis to stimulate cell proliferation but has relatively little effect on peripheral counts. IL-3 has been molecularly coupled to other growth factors, including GM-CSF, G-CSF, and TPO, to produce hybrid molecules that are under investigation. IL-11 is an early-acting factor that elevates blood platelets.(33) Stem cell factor (SCF)(34) and fms-like tyrosine kinase 3 (FLT-3) lig- and(35) are other early-acting factors under investigation. Macrophage colony-stimulating factor (M-CSF) is a selective factor for monocytes and macrophage formation,(36) and IL-5 is a similar selective factor for the generation of eosinophils.(37)
It is presumed that normally, hematopoietic cell formation is governed by combinations of factors, released in a cascade, that closely coordinate the development of these cells. The details of how this process occurs, however, are not yet clear. There are also numerous laboratory and clinical studies of combinations of factors, but the utility of using multiple growth factors therapeutically is not yet proved.
DYNAMICS OF HEMATOPOIESIS
The best estimate is that it takes 10 to 14 days for an event affecting the earliest stage of blood cell formation to be reflected in the peripheral blood count.(38) This time lag accounts for many clinical observations, such as the delay between time of infusion of stem cells and the rise in blood neutrophils, platelets, and erythrocytes with hematopoietic transplantation.
In the marrow, the blood elements develop in two phases, the proliferative and the maturational phases. During cell proliferation, the precursors of blood cells undergo cell division; normally, mitoses occur in the later stage of development at about 18- to 24-hour intervals. In the maturational phase, cell division ceases, but final features are added before the cells enter the blood. For instance, during this phase, erythrocytes normally lose all of their nuclear material, acquire their biconcave shape, and develop their final content of enzymes necessary for maintaining the biconcave shape and resisting destruction by oxidative stress.(17) During the proliferative phase, neutrophils acquire most of their granules--known as the primary, secondary, and tertiary granules--which are necessary for their microbiocidal activities.(38) During maturation, their nuclear chromatin condenses, the glycogen content of the cytoplasm increases, and the surface properties governing the circulation, adherence, and migration to tissues are added. Monocytes and eosinophils follow similar developmental patterns. Late in maturation, marrow neutrophils are mature enough to be readily released and functional; at this stage, they are regarded as being in the marrow neutrophil reserves. Quantitatively, this neutrophil pool is substantially larger--probably five to 10 times larger--than the total circulating supply of neutrophils.(38)
Platelets form from the cytoplasm of megakaryocytes, which are also derived from hematopoietic stem cells.(39) Megakaryocytes undergo reduplication of their nuclear chromatin without cell division, leading to production of extremely large cells. Platelets form from the breaking apart of the cytoplasm of the fully mature megakaryocytes or from filopodia. When marrow damage occurs from chemotherapeutic agents and after hematopoietic transplantation, the megakaryocytes are often the slowest cells to recover and thrombocytopenia is often the last cytopenia to resolve.
There are also important differences in the dynamics or kinetics of erythrocytes, platelets, and leukocytes in the blood. For instance, neutrophils turn over rapidly in the blood, with a blood half-life of six to eight hours, and essentially a new blood population of neutrophils is formed every 24 hours.(38)
Erythrocytes last the longest by far: the normal life span is about 100 days in the dog and human, about 150 days in horses, cattle, and sheep, and about 80 days in cats.(l7) These differences partially explain why neutrophils and red cells and their precursors are the predominant marrow cells, whereas erythrocytes far outnumber neutrophils in the blood. Similarly, the short half-life and high turnover rate of neutrophils explains why neutropenia is the most frequent hematologic consequence when bone marrow is damaged by drugs or radiation. Finally, transfusion of erythrocytes and platelets is feasible because of their relatively long life span, whereas the short life span of neutrophils has greatly impeded efforts to develop neutrophil transfusion therapy.
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