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122 Part II Cellular Basis of Hematology

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OLCs. Conversely, inhibition of the osteoclasts leads to reduced Hypoxia
HSPC and progenitor egress.
The niche for quiescent HSPCs is thought to be hypoxic. These
observations are based on studies that use an intracellular marker of
Bone Marrow Macrophages reducing equivalents, pimonidazole, or direct measurement of oxygen
tension in the BM using intravital microscopy. By direct measure-
During experiments investigating the mechanisms of G-CSF–induced ment of oxygen, it is apparent that the absolute level of PO 2 in the
HSPC mobilization, it was noted that in addition to previously BM is quite low (<32 mmHg) and reduces to ~10 mmHg deeper
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reported reduction in the endosteal OLCs, BM macrophages were into the marrow space. The vascular network in the marrow is
also decreased in number. In vivo depletion of this cell population extensive and it appears that the low PO 2 levels reflect high consump-
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by clodronate administration produced the same result. It therefore tion by the action of hematopoiesis.
appears that macrophages play a critical role in supporting the OLC Hypoxia is associated with upregulation of stromal cell–derived
niche compartment. A similar role for the macrophages, but with factor 1 (SDF-1) expression in the endosteal region and HSPC traffic
low
regard to supporting nestin-GFP MSCs, has been suggested by to the BM; in contrast, hyperbaric oxygen (exposure to 100% oxygen
another study that used clodronate-mediated macrophage ablation. under increased atmospheric pressure) mobilizes HSPC and progeni-
Again, this resulted in HSPC egress from the BM, perhaps through tors away from the BM. Hypoxic responses in the HSPCs are medi-
low
the effect on nestin-GFP , which showed a marked downregulation ated through a family of hypoxia-inducible factors (HIFs). The
of genes responsible for HSPC retention in the niche (see earlier best-studied species of HIFs is HIF1-α, which induces SDF-1 expres-
discussion). Thus, macrophages appear to function at a level of regu- sion and directs metabolic circuits within HSPCs toward anaerobic
low
lation upstream of OLCs and nestin-GFP cells. metabolism. HIF1-α also stimulates secretion of VEGF, thereby
promoting bone formation and angiogenesis. HIF1-α affects HSPC
function, as HIF1-α deletion from HSPCs leads to HSPC
Megakaryocytes exhaustion.
What is a physiologic role of hypoxia? Firstly, hypoxia is believed
Megakaryocytes are the most recent addition to a growing list of to protect HSPCs in the niche from oxidative stress. Indeed, HSPCs
niche cell types and appear to be directly involved in regulation of within the niche contain a lower level of reactive oxygen species.
HSPC quiescence, as shown by cell deletion studies. This effect is Moreover, it appears that hypoxic conditions are beneficial for the
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mediated by CXCL4, also known as platelet factor 4, and TGF-β. HSPC function because culturing human BM HSPCs under lower
Overall, only 20% of immunophenotypically defined HSPCs are oxygen tension leads to an increase in their ability to engraft and
associated with megakaryocytes, and were spatially distinct from repopulate nonobese diabetic/severe combined immune deficient
HSPCs that are located near the arterioles, suggesting the existence (NOD/SCI) mice. Finally, hypoxia may also protect the HSPC
of specific megakaryocytic niche. The above findings also illustrate a pool from exhaustion by promoting cell cycle quiescence. Of note,
feedback mechanism, by which mature hematopoietic cells may regu- when radiation or cytotoxic chemotherapy are used, the PO 2 levels in
late HSPC number. marrow go dramatically higher, raising the question of whether PO 2
levels participate in the hematopoietic regenerative response to injury.

EXTRINSIC REGULATION OF THE HEMATOPOIETIC STEM LYMPHOID NICHES
CELL NICHE
BM is the site of B-cell lymphopoiesis. Several cell types involved in
Sympathetic Innervation the HSPC niche also participate in formation of the lymphoid niches.
Interestingly, B-cell niches correspond to maturation stages of the
Sensory and autonomic innervation of the BM is critical for its ability lymphoid cells: OLCs, osteoclasts, and CAR cells are necessary for
to respond to hematopoietic stress. Increase in sympathetic tone the less mature stages of development, whereas interleukin-7 (IL-7)
promotes HSPC mobilization and downregulates the components of secreting cells and sinusoidal endothelial cells are important for more
the endosteal niche, mainly through the activation of β 2 -adrenergic differentiated cells. These observations come from targeted deletion
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receptor. Nestin-positive cells express both β 2 and β 3 receptors of each supporting cell population using genetic means and analyzing
and act as another mediator between sympathetic signaling and the effect on B-cell homeostasis. For example, deletion of the OLCs
HSPCs, again facilitating egress from the BM. In a mouse model leads to a considerable decrease in pre–pro and pro-B cells. This
of streptozotocin-induced diabetes, diabetic autonomic dysfunction process appears to be mediated by the heterotrimeric G protein α
was shown to disrupt this regulatory circuit, alter the function of subunit because its deletion in the OLCs leads to 60% decrease in
Nestin-positive cells, and lead to impaired G-CSF-induced HSPC the percentage of B-cell precursors in the BM. A similar phenotype
mobilization, providing a biologic explanation for a higher frequency is seen upon deletion of CAR cells.
of peripheral blood stem cell mobilization failure in diabetic patients. Naive recirculating B and T cells are located in the perisinusoidal
The sympathetic nervous system is also involved in regulation of space and colocalize with dendritic cells, which are thought to deliver
HSPC egress from the BM as governed by circadian rhythms, a supportive signals, because as their deletion leads to significant
remarkable discovery based on a chance observation that continuous decrease in B-cell number and reduction in IgM production after
exposure to light (because of a broken light switch in the animal immunization.
house) significantly altered the number of HSPCs mobilized after Plasma cells are the product of terminal differentiation of B cells
G-CSF administration. Thus, sympathetic niche innervation is after antigen exposure. In vitro and in vivo studies showed that
essential for relaying and integrating extrinsic signals, and acts as plasma cells receive multiple extrinsic survival signals, including
a responsive and finely tuned tool, which regulates HSPC traffic CXCL12, IL-6, BAFF (B-cell activating factor of the TNF family),
between peripheral blood and the BM. and APRIL (a proliferation-inducing ligand), which may account for
Not only sympathetic nerves, but also the glial cells that surround their longevity. Mice deficient in CXCR4 displayed impaired homing
them can regulate HSPC behavior in the marrow, as exemplified of plasmablasts, illustrating the involvement of CXCL12–CXCR4
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by the studies of nonmyelinating Schwann cells. These cells axis in plasma cell trafficking. Eosinophil- and megakaryocyte-derived
ensheathe sympathetic nerves, come into contact with HSPCs and are APRIL and BAFF appear to regulate the number of plasma cells,
responsible for activation of the latent form of TGF-β. Denervation which is greatly reduced upon eosinophil or megakaryocyte deletion.
experiments lead to significant reduction in the number of glial cells The majority of long-lived memory T cells reside in the BM and
and associated increase in HSPC cycling. appear to require a close contact with IL-7 secreting stromal cells to