Section

Hematopoiesis—the production of blood cells—is a tightly regulated system, exquisitely responsive to functional demands including infection, allergic reaction, immune challenge, hemorrhage, inflammation, and hypoxia. Beyond the production of blood cells, the integrity of the circulatory system also requires platelets, growth factors, and coagulation factors. Cells in the circulatory system sustain life by delivering oxygen and nutrients to tissue, clearing waste and pathogens, and recruiting humoral and cellular host defenses in a timely manner.

Hematology deals with the study of blood cells (e.g., erythrocytes, leukocytes, and platelets) and proteins in the circula tory system. Studies have shown that common blood disorders such as anemia, leukocytosis, and bleeding are indirect consequences of infection, inflammation, malnutrition, and malignancy. While hema-tologic malignancies could produce more severe bleeding, their prevalence is less common than blood disorders.

Advances in recombinant DNA technology have permitted cloning and production of growth factors and blood coagulation factors for the management of hematologic disorders. Several proteins can act on the growth and maintenance of each type of blood precursor cell. Each growth factor may elicit differentiation of a blood cell precursor into many distinct types of

Biotechnology and Biopharmaceuticals, by Rodney J. Y. Ho and Milo Gibaldi ISBN 0-471-20690-3 Copyright © 2003 by John Wiley & Sons, Inc.

leukocytes. Therefore it is important to understand the fundamental physiological principles of blood cells and growth factors. There follows a brief review of hematology with an emphasis on the therapeutic use of hematopoietic and coagulation factors.

Hematopoietic development of blood cells begins mainly in the spleen and liver of the fetus during early pregnancy. By the seventh month, however, the marrow of a fetus becomes the primary site of blood cell formation [1]. During childhood, the marrow of the central axial skeleton such as the pelvis, spinal cord, and ribs, and of the extremities, such as the wrist and ankle, provides the key site of hematopoiesis. Hematopoiesis at the periphery (also known as extramedullary hematopoiesis) slowly decreases with age. Chronic administration of hematopoietic growth factors can reverse this decline. Severe hemolytic anemia and hematopoietic malignancies can also reverse the process.

Production of blood cells in bone marrow of the central axial skeleton is referred to as medullary hematopoiesis. Hematopoietic tissue in adult bone marrow is well perfused and contains fat cells (adipocytes), and various types of blood and blood precursor cells encased within a protein matrix. Fibroblast, stromal and endothelial cells within bone marrow, serve as sources of matrix proteins as well as a factory for growth factors and chemokines that regulate blood cell production and release matured cells into the circulation [2,3]. Chemokines act as signal lamps for trafficking of lymphocytes in and out of lymphoid tissues. Erythroblasts, neutro-phils, lymphoblasts, macrophages, mega-karyocytes, and pluripotent stem cells are also found within the calcified lattice crisscrossing the marrow space.

All hematopoietic cells are believed to derive from a common precursor, hema-

topoietic stem cells [4]. Although stem cells constitute only about 0.05% of bone marrow, this population is maintained through a self-renewal system. Pluripotent stem cells undergo irreversible differentiation into daughter cells that are committed to lineages of unique hematopoietic cell types (Figure 6.1) [5]. While the mechanisms of early stages of lineage commitment by bone marrow to a particular type of blood cell remain elusive, the late stage of this process (differentiation and maturation process) is driven by hematopoietic growth factors. Many of these growth factors are now cloned and recombinant proteins are available for biologic and therapeutic studies. Some have had significant impact in treating hematologic disorders and managing neutropenia-associated infections.

Some hematopoietic growth factors exhibit overlapping specificities for cells of different lineages, particularly in the early stages of differentiation [6]. Studies with recombinant hematopoietic growth factors—including erythropoietin (EPO), thrombopoietin (TPO), granulocyte colony-stimulating factor (CSF), and macrophage colony-stimulating factor (M-CSF)—have shown that many of these proteins exhibit lineage-specific effects during the later stages of cell differentiation. In other words, lineage-specific growth factors tend to act on maturation and deployment of a given type of blood cell.

The cellular source and functional activity of hematopoietic growth factors are listed in Table 6.1. While the names "colony-stimulating factor," "-poietin," and "interleukin" are not obvious, they can be rationalized. The suffix "-poietin" as in erythropoietin and thrombopoietin derives from the Greek term poieis, meaning "to make." Colony-stimulating factors are so named because of their ability to stimulate target cells to divide and grow into colonies of cells in culture. The proteins produced by leukocytes that act on neighboring leukocytes are called interleukins. While most

Myeloid Stem Cell

IL-3 EPO

CFU-Mega

IL-3 IL-11

GM-CSF IL-3

GM-CSF IL-3

Proerythroblast Megakaryocyte Monoblast

Myeloblast

GM-CSF M-CSF

Eosinophilic Myeloblast

Pluripotent Stem Cell

IL-1 IL-6 SCF FLT-3L

GM-CSF IL-3

GM-CSF G-CSF

Red Blood Cell Platelets

Monocyte

Neutrophil

Eosinophil

GM-CSF IL-5

sophi elobl

Basophilic Myeloblast

Basophil

Prothymocyte

B Lymphoblast T Lymphoblast

Antigen-Driven

B Lymphocyte T Lymphocyte

Figure 6.1. The pathways used and hematopoietic growth factors required by pluripotient stem cells in bone marrow to differentiate and develop into distinct type of blood cells. The abbreviations of hematopoietic growth factors are found in Table 6.1.

Pre-B Cell

interleukins support leukocyte growth or lymphocytopoiesis, many also exhibit broad or pleotropic effects on cells of many lineages.

While it is assumed that hematopoietic cell formation is regulated through a concerted and coordinated release of factors in a cascade and series of reactions, the details of how such processes occur remain elusive. It is clear, however, that there are important differences in the growth and turnover (or removal) rates of blood cells. A new population of neutrophils is generated every 24 hours to replace aging cells, which are cleared with a half-life of about 6 to 8 hours [7]. On the other hand, ery-throcytes exhibit a well-documented life span of about 100 days. Despite the differences in capacity, turnover rate, and functions of blood cells, overall, hematopoiesis is tightly regulated to provide sufficient, but not excessive, numbers of different types of blood cells that are needed to sustain life.

The existence of some blood cells, such as erythrocytes and platelets, with long lifespans make cell transfusion therapy practical. Cell transfusion therapy cannot be developed for short-lived cells such as neu-trophils with turnover rates of less than 8 hours. Fortunately, for neutrophils, colony stimulating factors can be used to recruit the needed number in blood within 24 hours after administration of these factors.

ITABLE 6.1. Hematopoietic growth factors: Cellular source of synthesis, molecular characteristics, and functions

Hematopoiesis Factor

Cell Source

Functions

Molecular Characteristics

Granulocyte colony-stimulating factor; G-CSF; filgrastim; lenograstim Granulocyte -macrophage colony-stimulating factor; GM-CSF; Leukine; sagramostatin Macrophage colony-stimulating factor; M-CSF; colony-stimulating factor-1 (CSF-1)

Interleukin-la and ß; IL-la and ILl-ß; endogenous pyrogen hemopoietin-1 T cell growth factor; IL-2; aldesleukin; Proleukin

Multi-colony-stimulating factor; mast cell growth factor; IL-3

B cell growth factor; T cell growth factor II; mast cell growth factor II; IL-4 Eosinophil differentiation factor; eosinophil colony-stimulating factor; IL-5

Monocytes, fibroblasts, endothelial cells

Monocytes, T cells, and fibroblasts

Macrophages, endothelial cells, fibroblasts

Monocytes, endothelial cells, keratinocytes

T cells, large granular lymphocytes or natural killer (NK) cells Activated T cells, NK cells

T lymphocytes

T lymphocytes

Stimulates formation and function of neutrophils

Stimulates formation and function of neutrophils, monocytes, and eosinophils

Stimulates formation and function of monocytes

Support proliferation of T, B, and other cells; at high dose, it induces fever and catabolism

T-cell proliferation, antitumor and antimicrobial effects; at high dose, it induces fever and capillary leak syndrome Proliferation of early hematopoietic cells

Proliferation of B and T cells; enhances cytotoxic T-cell activities

Stimulates eosinophil formation; stimulates T-cell and B-cell functions

18.8 kDa protein containing 175 aa

15.5-19.5 kDa protein containing 127 aa

18.5 kDa protein containing 159 aa that form a 37 kDa dimmer in its active form

17 kDa proteins containing 153 (ILl-a) and 159 (ILl-ß) aa in their mature form 15.5 kDa protein

17.5 kDa protein containing 133-134aa

14 kDa protein containing 130aa

32-34 kDa glycoprotein containing 155 aa for each unit of homodimer

CD c

B cell stimulatory factor II; hepatocyte stimulatory factor; IL-6

Lymphopoietin 1; pre-B cell growth factor; IL-7

Plasmacytoma-stimulating factor; IL-11; oprelvekin; Neumega

Natural killer cell-stimulating factor; IL-12

Erythropoietin; EPO; epoetin

Thrombopoietin; TPO; megakaryocyte growth and development factor (MGDF)

Leukemia inhibitory factor; LIF

Stem cell factor; kit ligand; steel factor; SCF

/ws-like tyrosine kinase 3; FLT-3 ligand; STK-1

B and T cells, monocytes, fibroblasts, endothelial cells

Lymphoid tissues

Fibroblasts, trophoblastic placental cells

B cells, macrophages

Endothelial cells of kidney glomerular tubules

Renal and endothelial cells, hepatocytes, fibroblasts

Monocytes and lymphocytes, stromal cells

Hepatocytes, endothelial cells

T-lymphocytes, stromal cells, fibroblasts

Promotes B cell differentiation

22-27 kDa protein with 184 aa in its mature form

Promotes growth of B and T cells

Proliferation of early hematopoietic cells; induces acute-phase protein synthesis

Stimulates T cell expansion and interferon-gamma release; the combination of both agents synergistically promotes early hematopoietic cell proliferation

Stimulates erythrocyte formation and release from marrow

Stimulates megakaryocyte proliferation and platelet formation

Stimulates hematopoeitic cell differentiation

Stimulates proliferation of early hematopoietic cells and mast cells

Stimulates early hematopoietic cell differentiation; increases blood dendrite cells

17 kDa protein containing 153 aa

19 kDa protein containing 177 aa

75 kDa heterodimeric protein containing 306 aa p40 and 197 aa p 35 subunits

30.4 kDa glycoprotein containing 166 aa; 18.4 kDa unglycosylated

18-70 kDa highly glycosylated protein containing 332 aa

100-110kDa glycoprotein containing 789 aa in its mature form

18.5 kDa protein containing 164 aa

160kDa protein with 993 aa

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