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Knockout Mouse Models of Cytokine Action in Hematopoietic Stem Cell Regulation

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Hematopoietic stem cells are rare pluripotential cells within the blood-forming organs that are responsible for the continued production of blood cells during life. The regulation of hematopoietic stem cells is a complex process involving self-renewal, survival and proliferation, lineage commitment and differentiation and is coordinated by diverse mechanisms including intrinsic cellular programming and external stimuli such as adhesive interactions with the micro-environmental stroma and the actions of cytokines. Cytokines are predominantly secreted proteins that interact with specific receptors at the surface of target cells. An important role for cytokines in regulating hematopoietic stem cells has been inferred from their actions on purified stem cell populations in vitro. Over recent years, analysis of genetically modified or mutant mouse models has confirmed and defined the physiological roles for specific cytokines in hematopoietic stem cell production and function.

Introduction

In hematopoiesis, the production of blood cells, diverse cellular processes including self-renewal, proliferation, lineage specification, differentiation and maturation, apoptosis and effector functions are orchestrated with the precision needed to maintain the narrow ranges typical of mature blood cell numbers at steady-state as well as the flexibility that allows for rapid cellular production, migration or function demanded by bleeding or infection. Blood is continuously regenerating, with production of new cells carefully balanced to replace expended cells. The regenerative capacity of hematopoiesis originates with hematopoietic stem cells, a pool of rare cells within which the potential to generate the entire blood cell system resides. Accordingly, a defining property of hematopoietic stem cells is the capacity to self-renew, which ensures the maintenance of the pluripotential pool; but they must also differentiate into the progressively more mature hematopoietic progenitor cells, precursor cells and the circulating, mature functional blood cells. These hematopoietic stem cell functions are regulated by many distinct inputs including inherent genetic programming as well as external influences, most notably that of cytokines and the adhesive interactions with micro-environmental or stromal elements.

Analysis of hematopoietic stem cell production, function and regulation is a challenging task, primarily due to the rarity of these cells within the hematopoietic organs. Advances in defining cell surface markers via which hematopoietic stem cells can be recognized and isolated have improved the purity of stem cell populations available for study in vitro or in vivo. Hematopoietic stem cells in the bone marrow of mice lack expression of a series of antigens characteristic of differentiated blood cell progeny and depletion of these cells from the marrow, usually by immuno-magnetic techniques, enriches for stem cells. Among these Lin- marrow cells, pluripotent hematopoietic stem cells can be found in populations expressing c-Kit, Sca-1 and Thy1.1.1 The vital dye rhodamine-123 (Rh) has also been used to fractionate stem cell populations, with cells having the greatest long-term hematopoietic reconstituting capacity typically found among Rhlo fractions.2,3 The AA4.1 antigen has also proven useful in the purification of murine hematopoietic cells from fetal liver.4

A number of different functional assays can be employed in hematopoietic stem cell analysis. In vitro assays in which cells with the capacity to form cobblestone areas, blast cell colonies or high-proliferative potential multi-lineage colonies can provide insight into the multi-potential hematopoietic compartment, and the capacity of cells to initiate long-term hematopoietic cultures has also been correlated with stem cell properties.5 The CFU-s assay is a widely employed in vivo technique, in which the capacity of hematopoietic cells to form colonies in the spleens of myeloablated recipients is assessed. when scored 8 days after transplantation, the CFU-s assay detects relatively immature but committed progenitors, while day 12 CFU-s represent more pluripotential cells. It is widely accepted that the definitive assay for hematopoietic stem cells is measurement of the capacity to repopulate the entire hematopoietic system in transplanted hosts in which endogenous hematopoiesis has been ablated by a lethal dose of irradiation. Donor contribution needs to be monitored for many months following transplantation and quantitative assessment of stem cell capacity can best be achieved in competitive reconstitution assays in which the repopulating ability of test stem cells is directly measured against a standard competing dose of coinjected stem cells.6 Hematopoietic stem cell self-renewal can be stringently assessed in serial transplant studies in which the contribution of donor stem cells to hematopoiesis in secondary and subsequent recipients is examined.

Classical genetics exploiting mouse strain differences has been employed successfully to identify loci in mice that control hematopoietic stem cell number and function.7-10 Mutagenesis experiments using agents such as N-ethyl-N-nitrosourea (ENU) have also successfully identified genes potentially controlling hematopoietic stem cell regulation11 and recent advances in genomics technologies should see increased genetic dissection of hematopoietic stem cell regulation. The use of homologous recombination in embryonic stem (ES) cells to generate mice with precise genomic alterations has also provided a powerful tool for dissecting the role of specific gene products in the physiological context of the whole organism.12 As in other biological systems, knockout mice, in which a gene is typically functionally ablated using homologous recombination technology, have accelerated discovery of the roles of individual molecules in hematopoietic stem cell regulation. Knockout mice have been a powerful weapon in dissecting the origin and specification of hematopoiesis during embryonic ontogeny,13-15 and have helped identify and define the roles of diverse proteins in hematopoietic stem cell control, including transcription factors (reviewed in 16), cell cycle regulators,17,18 cell adhesion and stromal molecules19-23 and small GTPases.24,25 This review will focus on the use of knockout mice and other mouse mutants in the dissection of cytokine control of hematopoietic stem cells in vivo.

Cytokines and Signal Transduction

Cytokines are predominantly secreted proteins that control the proliferation, survival, maturation and mature effector function of cells in most body tissues including the blood forming system. In general, cytokines tend be produced at several sites in the body and usually act locally on a relatively restricted set of target cells. Nevertheless, most cytokines that act on hematopoietic cells exhibit pleoitropic and redundant actions. with few exceptions, most hematopoietic lineages are controlled by multiple cytokines and any given cytokine acts within several lineages.26 Cytokines act by binding to specific protein receptors expressed on the surface of target cells, an interaction which triggers receptor aggregation and activation followed by recruitment and activation of signalling intermediates. Numerous cascades of protein activation are initiated and ultimately integrated by the cell, resulting in changes in gene expression and the appropriate biological response.

The majority of cytokines that control hematopoiesis belong to the cytokine subgroups that adopt a 4-α-helical bundle structural topology. In turn, the specific receptors for the majority of these cytokines belong to either the hematopoietin or the tyrosine kinase receptor subclasses.27 The hematopoietin receptors are characterised by shared extracellular domain motifs, including several pairs of cysteine residues, the spacing of which is highly conserved, and the hallmark pentapeptide sequence Trp-Ser-Xaa-Trp-Ser (where Xaa is any amino acid, (Fig. 1). Although the hematopoietin receptors lack intrinsic catalytic activity, associated Janus kinases (JAK) become active upon ligand binding and receptor aggregation. This leads to phosphorylation of receptor tyrosine residues, resulting in recruitment of additional signalling molecules, as well as the direct activation by JAKs of independent signalling cascades. The signal transducers and activators of transcription (STAT) family of proteins are important mediators of hematopoietin receptor signals that are recruited to the phosphorylated receptor and activated in this way. In addition, changes in lipid metabolism, calcium flux and activation of the Ras/ MAP kinase pathway are among the other signalling cascades triggered by ligand binding to hematopoietin receptors.28 Similar signal transduction pathways are activated by ligand binding to the tyrosine kinase receptors, although in this case the tyrosine phosphorylation process is typically triggered by ligand-induced aggregation and activation of the receptors' intrinsic tyrosine kinase activity29 (Fig. 1).

Figure 1. Cytokines and receptors in hematopoiesis.

Figure 1

Cytokines and receptors in hematopoiesis. The interaction of cytokines with their cell surface receptors characteristically induces receptor aggregation, activation and initiation of intracellular signal transduction. Receptors in the hematopoietin receptor (more...)

Although the current understanding of cytokine control of hematopoietic stem cells is relatively rudimentary, maintenance of hematopoietic stem cells appears to require input from multiple cytokines. Considerable information has been gleaned from studies in vitro that have examined the actions of cytokines in the survival and proliferation of hematopoietic stem cells purified from humans or mice. In very simple terms, stem cell factor (SCF), Flk2/Flt3 ligand (FL) and thrombopoietin (TPO) appear to have the most potent effects on stem cell survival, maintenance of pluripotentiality and expansion in vitro, but even these cytokines are relatively impotent alone, and perform effectively only in combinations. Other cytokines, particularly interleukin (IL)-3, granulocyte colony-stimulating factor (G-CSF) and cytokines that use the gp130 receptor also have activity on stem cells in vitro, particularly when SCF, FL and/or TPO are also present.30-34 Many of these cytokines, their receptors and signalling intermediates, have now been functionally inactivated in knockout mice. The hematopoietic stem cell phenotypes of these genetically modified mice, as well as other mutant mouse models, are beginning to define the key roles of cytokines in the physiology of hematopoietic stem cell regulation.

Knockout Mice and Cytokine Control of Hematopoietic Stem Cells

Stem Cell Factor (SCF)

In mice, SCF is encoded by the steel (Sl) locus, while its receptor, the c-Kit protein, is the product of the dominant white spotting locus (W). Several naturally occurring mutant alleles of Sl and W, that affect the production or function of SCF or c-Kit, have been studied extensively over many years. The null alleles, Sl and W, cause embryonic lethality associated with severe anemia, but viable alleles, such as Sld, which lack membrane-bound SCF, and W41 or Wv, in which point mutations impair c-Kit tyrosine kinase activity, have allowed the effects of SCF signalling deficiency to be examined in adult mice. In addition to the hematopoietic defects discussed below, SCF or c-Kit deficiency also typically results in abnormalities in germ cell development that often result in sterility, and in melanogenesis, resulting in un-pigmented skin.

Consistent with an indispensable role for SCF signalling in hematopoietic stem cell regulation, the numbers of CFU-s in the bone marrow of mice harbouring Sl or W mutant alleles are reduced, typically by fourfold in W/Wv mutant marrow. Moreover, in contrast to the readily observable colonies produced by wild-type CFU-s, W/Wv mutant CFU-s, which are unable to respond to SCF in irradiated hosts, are capable only of microscopic colony formation. In long term in vivo reconstitution assays, W/Wv mutant hematopoietic stem cells fail to compete effectively with wild-type cells. Indeed, the hematopoietic stem cell defect in W/Wv mutants is such that wild-type donor stem cells can rapidly dominate hematopoiesis in W/Wv recipients, even in the absence of irradiation or other conditioning. Presumably as a direct consequence of the stem cell defect, numbers of progenitor cells of most hematopoietic lineages are slightly reduced in W/Wv mutant mice. Mature cell deficiencies are also evident in erythroid and mast cells, reflecting an additional specific role for SCF in maturing cells of these lineages.35

Studies of fetal liver hematopoiesis, particularly in Sld/Sld or W41/W41 mice, suggest that near-normal numbers of CFU-s and transplantable hematopoietic stem cells develop in the absence of SCF signalling, but that once bone marrow hematopoiesis is established in the adult, the marked deficiencies that typify the absence of SCF or c-Kit have arisen.36,37 Cotransplantation studies with wild-type and W41/W41 fetal liver cells suggested that while the mutant stem cells have a near-equivalent initial reconstituting capacity relative to wild-type cells, they are less competitive for long-term maintenance of hematopoiesis in myeloablated hosts.37 These data, which suggest that SCF is more important in the maintenance of hematopoiesis rather than the initial production of mature progeny from hematopoietic stem cells, have been interpreted to suggest that SCF acts predominantly to promote hematopoietic stem cell self-renewal. They also imply that hematopoietic stem cells, and their regulation by cytokines such as SCF, are qualitatively different in fetal liver in comparison with bone marrow (see Discussion).

Flk2/Flt3 Ligand (FL)

The Flk2/Flt3 protein, a tyrosine kinase receptor, was originally cloned from purified primitive hematopoietic populations38,39 and, within the hematopoietic compartment, Flk2/Flt3 expression is largely restricted to progenitor and stem cells.40-42 Long-term reconstituting stem cells are heterogeneous for Flk2/Flt3 expression: although most of the reconstituting activity of murine Lin-Sca+Kit+ stem cells appears to reside in the Flk2/Flt3- fraction, populations of Flk2/Flt3+ cells can clearly be shown to have long-term reconstituting capacity.43 FL, the specific ligand for Flt3/Flk2, is expressed in most human and mouse tissues examined.44,45

Homologous recombination in embryonic stem (ES) cells has been used to generate mice lacking FL or the Flk2/Flt3 receptor. Mice lacking Flk2/Flt3 undergo ostensibly normal development and appear healthy as adults. Although numbers of myeloid progenitor cells and CFU-s were unaltered in Flk2/Flt3-deficient mice, competitive repopulation studies revealed significant hematopoietic stem cell deficiencies. Bone marrow from mutant mice exhibited an approximately 5-fold reduction in repopulating potential relative to wild-type marrow. In the repopulated hosts, the contribution of Flk2/Flt3-deficient bone marrow to each of the myeloid, T- and B-lymphoid lineages was deficient. In addition to these defects within the hematopoietic stem cell compartment, anomalies were also observed specifically within the B-lymphoid lineage. B220+ cells were under-represented in Flk2/Flt3-deficient marrow, pro-B and preB cells appeared smaller than their normal counterparts, and B-lymphoid progenitor cells, assayed by IL-7-dependent in vitro colony formation, were 10-fold fewer in mutant than wild-type marrow.46

Mice lacking FL also develop into healthy adults. As observed in Flk2/Flt3-deficient mice, numbers of B-lymphoid progenitors were significantly reduced. However, in contrast to Flk2/Flt3-deficient mice, mice lacking FL exhibited reduced lymphoid cellularity in the peripheral blood, reduced bone marrow cellularity involving both immature lymphoid and myeloid cells, and fewer lymphocytes in the spleen and lymph nodes. The frequency of myeloid progenitor cells in FL-deficient bone marrow was similar to that observed in wild-type mice, but absolute progenitor cell numbers were reduced approximately 2-fold in the mutant mice. Consistent with a vital role in hematopoietic stem cell regulation, the absolute number of femoral CFU-s was reduced by 40% in FL-deficient mice relative to wild-type controls. Mice lacking FL also exhibit deficiencies in dendritic and natural killer cell populations.47 The data from the ligand and receptor knockout mice are consistent in supporting a physiologically critical role for FL in hematopoietic stem cell regulation. However, the basis for the discrepancies in mature blood cell numbers and cellularity of hematopoietic organs in FL- and Flk2/Flt3-deficient mice remains unresolved. There is little evidence that other cross-reactive ligands or receptors exist, that might account for this discrepancy. Thus, while strain differences have been proposed as the explanation for these inconsistencies in mature cell numbers, further data will be needed to fully clarify this issue.

Interestingly, flk2/flt3-/- W/Wv double mutant mice, which are deficient in both SCF and FL signalling, show exacerbated hematopoietic defects and fail to survive beyond six weeks of age. In vitro colony assays revealed similar frequencies of myeloid progenitor cells in double mutant bone marrow to that observed in the single knockout mice. However, marrow cellularity was significantly reduced resulting in an overall reduction of 8-fold in total progenitor cell numbers. B-lymphoid colony-forming cells were reduced 50-fold.46 Although not directly tested in stem cell assays, these data imply that the physiologically indispensable roles of SCF and FL signalling are nonredundant (see Discussion).

Thrombopoietin (TPO)

TPO is the major physiological regulator of platelet production that is produced primarily in the liver. The interaction of TPO with its receptor, the c-Mpl protein, drives production and maturation of megakaryocytes and their progenitors to ensure appropriate numbers of circulating platelets.48 The importance of TPO signalling in platelet production became clearly evident upon the generation of mice lacking TPO or c-Mpl. These mice exhibit indistinguishable phenotypes characterised by dramatically reduced circulating platelet numbers and deficiencies in megakaryocyte and megakaryocyte progenitor cell production in hematopoietic tissues.49-51

Further analysis of TPO- and c-Mpl-deficient mice revealed that, in addition to defects in megakaryocytopoiesis, progenitor cells committed to other hematopoietic lineages were consistently 2-fold fewer in the bone marrow of mutant mice compared with wild-type controls.49,52 The observation that the progenitor cell deficiency was most severe among the least mature blast colony-forming cells,49 coupled with data suggesting that c-Mpl is expressed in hematopoietic populations enriched for primitive cells,53,54 prompted the speculation that the multi-lineage deficiency in hematopoietic progenitor cells in TPO- and c-Mpl-deficient mice was symptomatic of a defect within the stem cell compartment.

Indeed, the necessity for TPO signalling in hematopoietic stem cell regulation became evident when these cells were directly assayed from c-Mpl-deficient mice. The bone marrow of c-Mpl-deficient mice contains up to 10-fold fewer CFU-s that wild-type marrow. This defect reflects an intrinsic defect in mpl-/- CFU-s, since the microenvironment in c-Mpl-deficient mice demonstrated a normal capacity to support CFU-s transplanted from wild-type donors.55 In reconstitution assays in myeloablated hosts, bone marrow cells from mpl-/- mice failed to compete effectively with normal cells for long-term reconstitution of the hematopoietic system, even when the c-Mpl-deficient cells were present in 10-fold excess over wild-type cells in the graft. In serial transplant studies, contribution to hematopoiesis by mpl-/- stem cells, albeit meagre in primary recipients, was undetectable in secondary or tertiary transplant recipients.55 Similar studies comparing Lin-Sca+ cells from wild-type and c-Mpl-deficient mice yielded consistent results, with a seven-fold reduction in potency observed for the mutant cells in competitive long-term reconstitution assays.56 This study also demonstrated elegantly that the hematopoietic repopulating capacity of purified murine and human stem cells resides predominantly in the fraction that expresses c-Mpl.56

Together, these analyses demonstrate unequivocally a key physiological role for TPO in regulation of hematopoietic stem cells. The reconstitution studies cannot formally distinguish between defects in production, self-renewal, survival and/or appropriate commitment and differentiation of hematopoietic stem cells in the absence of TPO signalling. Nevertheless, the CFU-s data revealed that the reduced numbers of these cells in c-Mpl-deficient marrow was not accompanied by any significant anomalies in colony size or composition among those mpl-/- CFU-s that did develop.55 This observation implies that TPO is required for the production of normal numbers of CFU-s, but is dispensable for their subsequent function. It is not clear whether this model also holds for the role of TPO in regulating more definitive reconstituting stem cells, although the need for TPO in stem cell self-renewal is strongly implied by the very poor performance of mpl-/- marrow upon serial transplantation.

Granulocyte Colony-Stimulating Factor (G-CSF)

G-CSF is a glycoprotein produced by activated macrophages, bone marrow stromal cells, fibroblasts and endothelial cells. G-CSF is a relatively specific stimulus of granulocyte colony formation in vitro and stimulates proliferation at all stages of granulopoiesis upon administration in vivo. Increases of up to 15-fold in peripheral blood neutrophil count can be achieved with G-CSF in vivo and G-CSF is commonly used clinically in the treatment of neutropenias. The ability of G-CSF to mobilise heamtopoietic progenitor and stem cells into the circulation of treated animals has also revolutionised transplantation medicine, with the use of G-CSF-elicited peripheral blood cells supplanting bone marrow cells for transplantation.26 The G-CSF receptor (G-CSFR) is a member of the hematopoietin receptor family (Fig. 1) that is expressed on neutrophils and monocytes and has also been detected on multi-potential and myeloid progenitor cells.57

Mice specifically engineered through gene targeting to lack G-CSF are healthy and fertile, but exhibit a chronic neutropenia with circulating neutrophil levels only 20-30% of normal.58 In the bone marrow of G-CSF-deficient mice, the numbers of maturing granulocytic precursor cells are low, as are granulocyte- and granulocyte-macrophage progenitor cells. As expected, a similar phenotype resulted in mice lacking the G-CSF receptor.59 In addition to steady-state netropenia, gcsf-/- mice exhibited a significant deficiency in Listeria monocytogenes induced neutrophilia and monocytosis, resulting in poor infection control and increased mortality.60

The role of G-CSF signalling in the physiological regulation of hematopoietic stem cells has not been extensively pursued. However, in experiments in which irradiated mice were transplanted with mixtures of wild-type and G-CSFR-null cells to produce hematopoietic chimeras, it was observed that approximately 5-fold greater numbers of mutant marrow cells than coinjected wild type cells was required to achieve equal repopulation. This observation infers that mice lacking G-CSF signalling are deficient in hematopoietic stem cell numbers, or that stem cells require G-CSF for proper function in transplanted hosts.61 Thus, more detailed analysis of mice lacking G-CSF or its receptor is likely to define an important role for this cytokine in the physiology of hematopoietic stem cells.

Interleukin-6 Cytokine Family

The interleukin-6 (IL-6) family of cytokines includes IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM) and cardiotropin 1 (CT-1). Each of these cytokines assumes a 4-α-helical bundle structure and interacts with hetero-oligomeric receptors that are characterised by the shared usage of the gp130 receptor signalling chain62 (Fig. 2). This cytokine family contributes important and highly pleiotropic functions in immune regulation and hematopoiesis, as well as regulation of the neural, endocrine, osteogenic and cardiovascular systems.63

Figure 2. Receptor structure of IL-6 family cytokines implicated in hematopoietic stem cell regulation.

Figure 2

Receptor structure of IL-6 family cytokines implicated in hematopoietic stem cell regulation. The IL-6, LIF and IL-11 receptors are hetero-oligomeric structures containing individual cytokine-specific chains and the shared gp130 signalling chain. Members (more...)

Mice lacking the shared gp130 receptor chain fail to survive gestation and die between embryonic day 12.5 and birth with heart defects and anemia. The numbers of erythroid- and myeloid-committed progenitor cells were reduced in fetal livers of gp130-deficient mice and defects in hematopoietic stem cell regulation were suggested by a significant deficit in fetal liver CFU-s.64 To avoid this embryonic lethality and allow the examination of gp130 function in post-natal life, conditional knockout mice were generated. Using the creloxP system,65 an allele was created that allows for functional inactivation of gp130 upon treatment of mice with interferon (IFN) α, which induced widespread expression of cre recombinase from an independent IFN-inducible transgene and subsequent excision in multiple tissues of a loxP-flanked region of the gene encoding gp130. Post-natal inactivation of gp130 in hematopoietic cells was efficient, and revealed that the deficiencies in hematopoietic progenitor cells and CFU-s that were evident in the fetal livers of the original gp130-/- mice were reproduced in analysis of the bone marrow in this model of post-natal gp130 deficiency.66 These data suggest that signalling from the gp130 receptor chain contributes to hematopoietic stem cell regulation in both fetal liver and bone marrow hematopoiesis.

Insight into which of the individual IL-6 cytokine family members contributes to hematopoietic stem cell regulation has also been investigated using knockout mice. Mice lacking IL-6 develop apparently normally and grow into healthy adults. However, il6-/- mice exhibit a 3-fold reduction in bone marrow CFU-s and a 5-fold reduction in preCFU-s, defined in this study as cells in the bone marrow of primary marrow recipients that have the capacity to generate spleen colonies in secondary myeloablated hosts. In competitive long-term reconstitution studies, IL-6-deficient bone marrow competed effectively in primary recipients, suggesting relatively normal numbers of hematopoietic stem cells develop in the absence of this cytokine. However, upon serial transplantation into secondary and subsequent recipients, IL-6-deficient stem cells exhibited a progressive decrease in competitive reconstitution. Thus IL-6 appears to have a particular role in hematopoietic stem cell self-renewal, as well as effective production of more mature progeny such as CFU-s.

Mice lacking LIF also display significant hematopoietic stem cell deficiencies. CFU-s numbers were reduced by 90% in the spleen and 60% in the bone marrow, when compared with wild-type controls. Transplantation studies comparing survival and hematopoiesis in myeloablated mice receiving a CFU-s-equivalent dose of either wild-type of LIF-deficient bone marrow suggested that there was no inherent defect in stem cell potential. Survival of transplanted mice, as well as the numbers of CFU-s and hematopoietic progenitor cells in the spleens and marrow, was equivalent among recipients of wild-type and LIF-deficient cells.67 This observation was interpreted to indicate that LIF is an important component of the hematopoietic stem cell microenvironment that is necessary for the production and maintenance of stem cells rather than their subsequent function.

Hematopoiesis in mice lacking the specific IL-11 receptor α-chain has been examined extensively. CFU-s numbers and hematopoietic recovery from cytotoxic insult in IL-11Rα-deficient mice revealed no apparent defect.68 Thus, although definitive hematopoietic reconstitution studies have not been performed, there is currently no evidence that IL-11 is required for physiological regulation of stem cells.

Tumor Necrosis Factor Receptor (TNFR) and Fas

The TNF receptor family includes the two TNF receptors, p55 and p75, as well as the Fas protein and over twenty other cell surface molecules involved primarily in lympho-hematopoietic regulation. The actions of TNFα, which include protection against infection as well as pathological effects in septic shock, autoimmunity, inflammatory diseases and diabetes, appear to be predominantly mediated via p55, or the type I TNFR. The role of the type 2 TNFR, or p75, remains less well defined.69 The Fas protein is the cell surface receptor for Fas ligand (FasL), itself an integral membrane protein that can also be cleaved into soluble form, that is an important apoptotic effector molecule for cytotoxic T cells and NK cells.70 Fas and p55TNFR share structural similarities, most notably the presence of conserved cysteine-rich regions within the extracellular domain and an intracellular “death domain,” which links the activated receptors with intracellular apoptotic pathways71 (Fig. 1).

In young adult mice lacking p55TNFR, an mild elevation in myeloid progenitor cells was evident, but no difference in the competitive repopulating capacity of mutant stem cells was observed. In contrast, in comparison with age-matched wild-type mice, older mice lacking p55TNFR exhibited significantly elevated circulating white blood cells and increased bone marrow cellularity involving most mature marrow cell types as well as clonogenic progenitor cells. Paradoxically, while CFU-s numbers in the marrow of p55TNFR-deficient mice were mildly elevated, a 4-fold decrease in the activity of hematopoietic stem cells was indicated by competitive repopulation assays.72 Studies examining recipients of approximately equivalent numbers of stem cells (4-fold more bone marrow cells in mice receiving p55TNFR-deficient marrow) indicated that contribution to hematopoiesis from stem cells lacking p55TNFR declined more rapidly over time than that from wild-type cells. The capacity of p55TNFR-deficient hematopoietic stem cells was also significantly below that observed for wild-type cells in serial transplants in secondary and tertiary recipients.72 These data suggest that the both the numbers and self-renewal capacity of hematopoietic stem cells are deficient in ageing mice lacking p55TNFR. Based on these data, the authors of this study propose a model in which signals from p55TNFR affect the balance between self-renewal and differentiation into maturing progeny, with the latter favoured over the generation of new stem cells. Such a model might account for the unusual observation that age-related exhaustion of hematopoietic stem cells capacity is accompanied by elevated numbers of mature progeny in p55TNFR-deficient mice. Since TNFα is capable of inhibiting entry of hematopoietic stem cell into the cell cycle,73 it is feasible that in the absence of p55TNFR the signals maintaining stem cell quiescence are also diminished.

The lpr mutant mouse develops an age-dependent autoimmune disease that involves anomalies in both T- and B-lymphopoiesis and which exhibits similarities to human lupus. The lpr allele arose as a result of retro-transposon insertion into the locus encoding Fas, resulting in significantly reduced expression of the Fas receptor,74,75 In Fas-deficient lpr mice, numbers of splenic CFU-s are elevated relative to wild-type controls. This anomaly is evident as early as one month of age and becomes more striking with increasing age. The numbers of CFU-s in the bone marrow appeared not to be affected, although a transient increase in marrow CFU-s in newborn lpr mice was reported and numbers in the fetal livers of these mice were clearly elevated.76 While a role for Fas/FasL in hematopoietic stem cell regulation, similar to that observed for the related TNFR, is implied by these studies, analysis of the behaviour of lpr stem cells in reconstitution studies will be needed to confirm and more precisely define such a role.

Components of Cytokine Signal Transduction Pathways

The interaction of cytokines with their receptors triggers a multitude of signalling pathways that are ultimately integrated into the cellular response to cytokine stimulation. There is evidence to suggest that different signalling cascades may differentially promote distinct responses such as cytokine-induced proliferation and differentiation. Accordingly, it is of considerable interest to discover not only those cytokines that have important roles in hematopoietic stem cell regulation, but also the key signalling cascades that execute cytokine actions.

STAT5, a member of the signal transducer and activator of transcription family, is activated in cells stimulated by a diverse array of hematopoietic and other cytokines. Mice lacking either of the STAT5 isoforms, STAT5A or STAT5B, exhibit mild if any hematopoietic defects. However, double knockout mice display reduced T cell numbers, a reduction in hematopoietic progenitor cell numbers and deficient erythropoiesis.77,78 A significant reduction in the number of CFU-s and the size of the colonies they produced was evident in STAT5A/B double-deficient mice. While bone marrow and fetal liver cells from STAT5A/B double knockout mice were capable of reconstituting the hematopoietic compartment in myeloablated hosts, the capacity of hematopoietic stem cells from these animals to effectively compete with wild-type cells in a competitive repopulation assay was significantly diminished.79,80 Presumably, this phenotype reflects a role for STAT5 in the stem cell regulatory actions of a specific cytokine or cytokines. Since STAT5 is known to be activated by TPO, SCF and Flk2/Flt3 ligand,81-83 it is tempting to speculate that among the variety of intracellular signals triggered by these cytokines, the STAT5 pathway is specifically required for their actions on hematopoietic stem cells.

The Lnk protein is an SH2 domain-containing adaptor molecule that was originally identified as a link between the activated T cell receptor and multiple signalling cascades.84 More recently, Lnk has been implicated in negative regulation of SCF signalling. Upon SCF stimulation, the c-Kit protein phosphorylates and associates with Lnk, which is thought to selectively inhibit c-Kit-mediated proliferation by preventing activation of the MAP kinase cascade.85 Mice engineered to lack Lnk exhibit enhanced production of B cells, which has been attributed to hyper-responsiveness of B-lymphoid precursor cells to SCF.86 More recently lnk-/- mice have been used to demonstrate that Lnk is also a key negative regulator of hematopoietic stem cell regulation. Mice lacking Lnk contained twice the wild-type number of bone marrow CFU-s and produced stem cells capable of out-competing wild-type cells in competitive repopulation assays.85 The excess B-lymphopoiesis typical of Lnk deficiency was significantly ameliorated in lnk-/- W/+ mice, in which c-Kit expression is reduced. However, the capacity of lnk-/- W/+ bone marrow in competitive reconstitution assays appeared comparable to that of lnk-/-+/+ marrow.85 Since c-Kit expression is reduced rather that absent in lnk-/- W/+ mice, it cannot be excluded that these data reflect a lower threshold of response to c-Kit signalling by hematopoietic stem cells versus B-lymphoid precursors. Nevertheless, these studies infer the existence of important c-Kit-independent pathways that are controlled by Lnk in hematopoietic stem cells, possibly initiated by other cytokines.

Discussion

The analysis of mice in which spontaneous or engineered mutations prevent the appropriate expression of cytokines or their intracellular signals has unequivocally confirmed an indispensable physiological role for cytokine action in hematopoietic stem cell regulation. As might have been anticipated, cytokines appear to deliver both positive and negative physiological signals, and functions in different aspects of stem cell biology, including production and maintenance, self-renewal and the capacity to generate differentiated progeny have all been revealed in the specific phenotypes of mice lacking cytokines or their signal transducers that are detailed above and summarised in Table 1. To a significant degree, the cytokines implicated in hematopoietic stem cell regulation by virtue of their actions on purified stem cells in vitro have also proven to have a significant physiological role. Perhaps the most notable exception is IL-3. IL-3 is a potent stimulus for multi-potential progenitor cells in culture and has also been included in cytokine cocktails that support purified hematopoietic stem cells in vitro.87 Nevertheless, mice lacking IL-3 have apparently normal steady-state hematopoiesis88,89 with defects evident only in the contexts of delayed-type hypersensitivity and immunity to parasites.88,90 Moreover, the general reduction in hematopoietic progenitor cells that arises from stem cell defects in mpl-/- mice is not exacerbated by the loss of IL-3 in c-Mpl/IL-3 double mutant mice.89 However, examination of IL-3-deficient stem cells in CFU-s or definitive long-term reconstitution studies has not been reported, and future analyses of this kind may be rewarding.

Table 1. Phenotypes in mice lacking signals from selected cytokines implicated in hematopoietic stem cell regulation.

Table 1

Phenotypes in mice lacking signals from selected cytokines implicated in hematopoietic stem cell regulation.

An invariant theme that has emerged form the study of cytokine-deficient mice is that no single cytokine appears responsible for the entire capacity of the hematopoietic stem cell pool. For example, while substantial defects in the numbers or function of stem cells are clearly evident in mice lacking signals from TPO, SCF, FL, IL-6 or LIF, in each case sufficient stem cell capacity exists to support ongoing hematopoiesis. In most cases, despite the stem cell deficiency, relatively normal numbers of mature blood cells reach the hematopoietic organs and circulation, excepting, of course, in those lineages in which the missing cytokine also has a role in differentiated cells. This observation implies that, while compensatory mechanisms must exist to boost the production of mature cells from a sub-optimal immature cell pool, a reduced but ongoing level of hematopoietic stem cell activity is sustainable in the absence of individual cytokines. An obvious hypothesis, strongly supported by the observations that multiple cytokines are required to support hematopoietic stem cells in vitro, is that the wild-type level of stem cell production and function in vivo is dependent on the concerted action of multiple cytokines. When knockout mice lacking multiple cytokines have been generated to test this hypothesis, support for this model has emerged from mice unable to respond to both FL and SCF, in which stem cell deficiencies appear to be compounded relative to mice lacking FL or SCF signalling alone.46 However, in other examples, such as mice lacking both c-Mpl and IL-6 an exacerbation of stem cell defects is not apparent.91 It should also be noted that in each of these double knockout studies, hematopoietic stem cell function was not assessed by stringent long-term reconstitution assays. Thus, while these studies provide clear evidence for redundancy in the actions of cytokines in the physiological regulation of hematopoietic stem cells, the intricacies of this cytokine network remain to be fully elucidated. For example, whether hematopoietic stem cells defects in mice lacking a single cytokine reflect a reduced net signal common to all stem cells, or result from elimination of a subset of cells strictly dependent upon that cytokine, cannot yet be distinguished from the analyses to date.

The study of knockout mouse models also reinforces the notion that hematopoietic stem cells and the systems that regulate them are different in fetal and adult life. The analysis of W mutants suggest that stem cells in adult mice are more dependent on SCF signalling that their counterparts in fetal liver.37 Curiously, the deficiency in hematopoietic progenitor cells of multiple lineages that arises from the stem cell defect in adult mpl-/- bone marrow appears not to be present in the fetal livers of these mutants, suggesting that fetal and adult stem cells may also show a differential dependence on TPO signalling.49 These observations, which suggest a limited dependency of fetal hematopoietic stem cells on cytokines, might imply that stem cell production and function during embryonic development is driven largely by cytokine-independent or intrinsic developmental programming. Alternatively, the cytokine combinations that support hematopoietic stem cells in fetal life may be distinct from those utilised in the adult. Thus, as well as helping to define the physiological roles of cytokines in adult hematopoiesis, knockout mouse models of cytokine action will also provide valuable reagents to dissect regulatory pathways in hematopoietic stem cell ontogeny.

Acknowledgements

Work in the author's laboratory is supported by the National Health and Medical Research Council, Canberra, Australia, the National Institutes of Health, Bethesda, Grants No HL62275 and CA22556, and the Australian Government Cooperative Research Centres Program. I am grateful to Professor Donald Metcalf for helpful discussions and comments on the manuscript.

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