Trafficking of primitive hematopoietic cells

The migratory behavior characteristic of primitive hemat-opoietic cells is an area of intense research because of its relationship to bone marrow transplantation. Trafficking of HSCs can be divided into the components of homing, retention and engraftment. 'Homing' describes the tendency of cells to arrive at a particular environment, while 'retention' is their ability to remain in such an environment after arrival. Lastly, 'engraftment' reflects the ability of cells to divide and form functional progeny in a given microenvironment. Much has

Table 3.1 Factors affecting hematopoietic control.

Growth factor

Growth factor receptor

Produced by

Bioactivity

Deficient states

Erythropoiesis

EPO (erythropoietin)

EPO-R

Adult kidney Liver during development

Stimulates clonal growth of CFU-E and BFU-E subsets Suppresses erythroid progenitor cell apoptosis Induces bone marrow release of reticulocytes Induces erythroid globin synthesis

Anemia

SF (steel factor), kit ligand, mast cell growth factor

c-kit (CD117)

Fibroblasts Endothelial cells Bone marrow stroma

Promotes proliferation and differentiation of pre-CFC cells Acts synergistically with IL-3, GM-CSF and TPO to support growth of CFU-GEMM, BFU-E, and CFU-Mk Expansion of committed progenitor cells in vivo Stimulates mast cell hyperplasia, degranulation, and IgE-dependent mediator release

Anemia Mast cell deficiency

IGF-1 (insulin-like growth factor, somatomedin C)

IGF-1R

Liver

Induces DNA synthesis and has anti-apoptotic effects in erythroid progenitors

Simulates erythroid colony growth in the absence of EPO at high doses

Growth retardation, neurologic defects, homozygous deficient lethal

Granulopoiesis

Granulopoiesis

G-CSF (granulocyte G-CSFR

colony-stimulating factor)

GM-CSF (granulo- GM-CSFR

cyte-macrophage colony-stimulating factor)

M-CSF (macrophage c-fms colony-stimulating factor)

Thrombopoietin c-mpl

Monocytes, macrophages, endothelial cells, fibroblasts

Stimulates growth of progenitors committed to neu-trophil differentiation Activates neutrophil phagocytosis Stimulates quiescent HPCs to enter G,-S Stimulates mobilization of HSCs and HPCs from bone marrow to periphery

Mast cells, T lym- Stimulates multilineage hematopoietic progenitor cells phocytes, endothelial Stimulates BFU-E and granulocyte, macrophage, and cells, fibroblasts, thymic eosinophil colony growth epithelial cells

Monocytes, macrophages, fibroblasts, epithelial cells, vascular endothelium, osteoblasts

Bone marrow stroma, spleen, renal tubule, liver, muscle, brain

Induces monocyte/macrophage growth and differentiation and activation

IL-5

IL-5R

IL-11R

T lymphocytes

Fibroblasts, bone marrow stroma

Stimulates in vitro growth of CFU-Mk, megakaryocytes and platelets

Stimulates clonal growth of individual CD34+38- cells Synergizes with SF, IL-3 and FL

Primes response to platelet activators ADP, epinephrine and thrombin but no effect on aggregation

Stimulates eosinophil production and activation Activates cytotoxic T cells Induces immunoglobulin secretion

Acts synergistically with IL-3 or SF to stimulate the clonal growth of erythroid (BFU-E and CFU-E) and primitive megakaryocytic (BFU-Mk) progenitors Shortens duration of G0 of HPCs Quickens hematopoietic recovery after chemotherapy and radiation

Neutropenia, failure to develop neutrophilic leukocytosis in response to infection

Susceptibility to infections caused by obligate intracellular organisms

Macrophage and osteoclast deficiency, hematopoietic failure

Thrombocytopenia

Inability to mount eosinophilic response

No hematological defect

Continued

Table 3.1 (Continued.)

Growth factor

Growth factor

receptor

Produced by

Bioactivity

Deficient states

Lymphopoiesis

IL-7

IL-7R

Bone marrow

Induces clonal growth of pre-B cells

B- and T-cell lym-

stroma, spleen,

Induces growth of pre-T cells

phopenia

thymus

IL-2

IL-2R

T lymphocytes

Induces proliferation and activation of T cells, B cells and

Fatal immunopro-

NK cells

liferative disorder,

loss of self-toler-

ance

IL-15

IL-15R

Monocytes,

Induces proliferation and activation of T cells, B cells and

macrophages,

NK cells

epithelial cells,

skeletal muscle

cells, bone marrow

and thymic stroma

IL-4

IL-4R

T lymphocytes

Induces proliferation of activated B cells

Defective T helper

Inhibits IL-2-stimulated proliferation of B cells

cell responses

Induces T cell proliferation

IL-10

Inhibits monocyte/macrophage dependent synthesis of

Th1- and Th2-derived cytokines

Early-acting factors

IL-3

IL-3R

T lymphocytes,

Stimulates multilineage colony growth and growth of

No hematopoietic

mast cells

primitive cell lines with multilineage potential

defect in steady

Stimulates BFU-E proliferation

state, deficient

delayed-type

hypersensitivity

FLT3-ligand (FL)

FLT-3R, flk2

Most tissues,

Weak colony-stimulating activity alone but synergizes with

Reduction in pro-B

including spleen,

IL-3, GM-CSF, SF, IL-11, IL-6, G-CSF, IL-7, and others

cells, pre-B cells,

lung, stromal cells,

Augments retroviral transduction of HSCs when added to

B-cell colony-

peripheral blood

cytokine cocktails

forming potential,

mononuclear cells

Mobilizes HSCs to periphery weakly alone but adds greatly

reduced repopu-

to G-CSF

lating capacity of

stem cells

IL-9 (T-cell growth

IL-9R

T lymphocytes

Stimulates growth of BFU-E when combined with EPO

factor)

Stimulates clonal growth of fetal CFU-Mix and CFU-GM

IL-6

IL-6R

Macrophages,

Synergistic with IL-3 for CFU-GEMM colony growth

Reduced HSC and

endothelial cells,

Synergistic with IL-4 in inducing T cell proliferation and

progenitor cell

fibroblasts, T

colony growth

survival, reduced

lymphocytes

Synergistic with M-CSF in macrophage colony growth

T cell numbers, re-

Synergistic with GM-CSF in granulocyte colony growth

duced proliferation

Co-induces differentiation of B cells

and maturation

of erythroid and

myeloid cells

BFU-E, burst-forming unit—erythroid; CFU-mix, colony forming unit—mix; CFU-Mk, colony forming unit—megakaryocyte; CFU-GM, colony

forming unit—granulocyte/macrophage

CFU-GEMM, colony forming unit granulocyte, erythroid, monocyte, megakaryocyte.

been learned about trafficking from the ontogeny of mouse and human HSCs.

Hematopoietic ontogeny

In both humans and mice, hematopoiesis occurs sequentially in distinct anatomical locations during development. These shifts in location are accompanied by changes in the functional status of the stem cells and reflect the changing needs of the developing organism. These are relevant for adult hemat-opoiesis since they offer insight into how the blood production process can be located in different places with distinct regulation.

There are essentially four sites of blood cell formation recognized in mammalian development, and these are best defined in the mouse. At about embryonic day 7.5 (E7.5), blood and endothelial progenitors emerge in the extra-embryonic yolk sac blood islands. The yolk sac supports the generation of primitive hematopoietic cells, which are primarily composed of nucleated erythrocytes. More sustained or definitive hematopoiesis may derive from the yolk sac, but this remains controversial. However, the aorta-gonadal-mesonephros (AGM) region has been clearly identified as the first site of definitive hematopoiesis in both the mouse (E8.5) and the human. It is not clear if the yolk sac seeds the AGM region or if the hematopoietic cells arise there de novo, but by E10 in the mouse the fetal liver assumes the primary role of cell production. By E14 in the mouse and the second trimester of human gestation, the bone marrow becomes populated with HSCs and it takes over blood cell production, along with the spleen and thymus. The spleen remains a more active hematopoietic organ in the mouse than in the human.

The transition in the location of hematopoiesis is roughly associated with changes in HSC function. Primitive hematopoiesis and definitive hematopoiesis in the AGM is dominated by the production of red blood cells and stem cells. Because the organism and its vascular supply become more complex when the fetal liver becomes a hematopoietic site, platelet production is added to the robust production of erythrocytes and stem cells. In keeping with the shifting needs of the organism, by late gestation a full spectrum of innate and adaptive immune system cells is added to the production repertoire. Stem cell proliferation begins to decrease and eventually reaches a state of relative quiescence shortly after gestation.

Homing and engraftment of HSCs following infusion

Despite the use of HSC transplantation for over three decades, the exact mechanisms whereby bone marrow cells home to the bone marrow are not fully understood. Other than lectins, no adhesion receptors have been identified that are exclusively present on HSCs. Furthermore, no adhesion ligands, other than hemonectin, have been identified that are exclusively present in the bone marrow microenvironment.

When first infused, HSCs lodge in the microvasculature of the lung and liver; they then colonize the bone marrow, first passing through marrow sinusoids, migrating through the extracellular space of the bone marrow, and ultimately land in the stem cell niches. Passage through endothelial barriers at first requires tethering, through endothelium-expressed addressins that bind hematopoietic cell selectins, and this is followed by firm attachment mediated by integrins.

Selectins are receptors expressed on hematopoietic cells (L- and P-selectins) and endothelium (E- and P-selectins). They have long extracellular domains containing an amino-terminal Ca2+-binding domain, an epidermal growth factor domain, and a series of consensus repeats similar to those present in complement regulatory molecules. Ligands for selectins are sialylated fucosylglucoconjugates present on endothelium, termed 'addressins'. L-selectin is present on CD34+ hematopoietic progenitors while L-selectin and P-selectin are present on more mature myeloid and lymphoid cells. Tethering by selectins allows integrin-mediated adhesion to the endothelium. Integrins, a family of glycoproteins composed of a and (3 chains responsible for cell-extracellular matrix and cell-cell adhesion, provide not only firm attachment but also allow migration of hematopoietic cells through the endothe-lium and bone marrow extracellular space. The functional state of integrins is only loosely tied to their expression level and depends on ligand affinity modulation regulated by the ( subunit in response to cytokines and other stimuli.

The process of migration depends on the establishment of adhesion at the leading edge of the cell and simultaneous release at the trailing edge. The rate of migration depends on dynamic changes in the strength of the cell-ligand interactions, which is dictated by the number of receptors and their affinity state and the strength of the adhesion receptor-cy-toskeleton interactions. Cell-ligand interaction strength may also be modulated by cytokines. Thus, successful engraftment relies not only on the presence of several different adhesion receptors but also on their functional state and ability to facilitate both migration and adhesion.

Egress of HSCs from bone marrow under physiological conditions

The majority of primitive HSCs are resident within the bone marrow space under steady-state physiological conditions. However, a population of CD34+ cells capable of forming CFCs and LTC-ICs and capable of long-term repopulation may be found circulating in the peripheral blood and these may increase after physiological stressors such as exercise, stress and infection. Recent studies have suggested that a relatively large number of bone marrow-derived stem cells circulates during the course of a day and that these cells periodically transit back into an engraftable niche to establish hematopoiesis. Defining the processes involved is important in guiding new approaches to peripheral blood stem cell mobilization for transplantation.

Examining mice in which specific adhesion molecules have been deleted has revealed several key molecular determinants of stem cell localization in the bone marrow. Among these, the chemokine receptor CXCR4 has perhaps the most striking phenotype. In the absence of this receptor, stem cells fail to traffic from the fetal liver to the bone marrow. Partly because of these studies, others have defined that CXCR4 is relevant for the engraftment of transplanted stem cells and that the modulation of CXCR4 signaling can affect adult stem cell localization in the bone marrow versus peripheral blood. As described, the integrin and selectin families are also important molecular participants in stem cell location. For example, HSCs from animals that are heterozygous-deficient for P1 integrin cannot compete with wild-type cells for the colonization of hematopoietic organs. Pre-incubation of HSCs with a4 integrin antibodies prior to transplantation results in decreased bone marrow and increased peripheral recovery of cells, while the continued presence of a4 antibodies prevents engraftment. Evidence for selectin involvement has been demonstrated in animals deficient for single selectins or combinations of selectins. Endothelial P-selectin mediates leukocyte rolling in the absence of inflammation, while L-, P- and E-selectins contribute to leukocyte rolling in the setting of inflammation. L-selectin is important in lymphocyte homing. Transplantation studies performed in animals deficient in P- and E-selectins demonstrate severely decreased engraftment due to impaired homing, an effect that is further compromised by blocking vascular cell adhesion molecule-1 (VCAM-1).

Mature hematopoietic cells are thought to migrate from the marrow to the blood by similar mechanisms, though these are not well defined. One purported mechanism is a shift in expression from molecules thought to interact with stromal proteins to those that interact with endothelium. For example, myeloid progenitors express functional a4P1 and a5P1 integrins that act to ensure that these progenitors are retained in the bone marrow through interactions with VCAM and fibronectin. Mature neutrophils, in contrast, express P2 integrins that permit interaction with ligands, such as intercellular adhesion molecule (ICAM), expressed by endothelial cells. Mature neutrophils also express P1 integrins that permit interaction with collagen and laminin present in basal membranes, perhaps regulating a progressive shift in cell affinities for specific microenvironmental determinants that ultimately results in cell egress into the blood. Mobilization of murine

HSCs induced by cyclophosphamide or granulocyte colony-stimulating factor (Cy/G-CSF) is accompanied by changes in integrin expression levels and functional changes in homing, thus linking cellular localization with adhesion molecule receptor expression.

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