Volume 75, Issue 2, Pages 79-93 (August 2010)

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Abnormal immunity and stem/progenitor cells in acquired aplastic anemia

Accepted 9 December 2009. published online 04 January 2010.

Abstract

Acquired aplastic anemia (AA) is considered as an immune-mediated bone marrow failure syndrome, characterized by hypoplasia and pancytopenia with fatty bone marrow. Abnormal immunity is the major factor mediating the pathogenesis of acquired AA. Activated DCs might promote the polarization to Th1 cells, and activate CD8+ T cells. A variety of immune molecules including IFN-γ, TNF-α, MIP-1α and IL-2, 8, 12, 15, 17, 23, produced by them and stromal cells, compose a cytokine network to destruct stem/progenitor cells as well as hematopoietic stem/progenitor cells, mesenchymal stem cells (MSCs) and angioblasts/endothelial progenitor cells. Inversely, deficient MSCs, CD4+CD25+ T cells, NK cells, NKT cells and early hematopoietic growth factors diminish the capacity of immune regulation and the support of hematopoiesis. As a result, stem/progenitor cells are significantly impaired to be disabled cells with markedly deficient proliferation, differentiation, induced apoptosis and dysfunctional response to growth factor stimuli, together with rare normal ones. Although some patients can be ameliorated by stem-cell transplantation or immunosuppressive therapy, more effective and convenient therapies such as patient-specific pluripotent iPS cells based on definite pathogenesis are expected.

Keywords: Immunologic mechanism, Stem/progenitor cell, Acquired aplastic anemia

Article Outline

• Abstract

• 1. Introduction

• 2. Immunologic mechanisms

• 2.1. Immune tissues

• 2.1.1. Bone marrow

• 2.1.2. Thymus, spleen, lymph node

• 2.2. Immune cells

• 2.2.1. Innate immune cells

• 2.2.2. Adaptive immune cells

• 2.3. Immune molecules

• 3. Stem/progenitor cells and immune impairment

• 3.1. Hematopoietic stem/progenitor cells and immune impairment

• 3.2. Mesenchymal stem cells and immune impairment

• 3.3. Angioblasts/endothelial progenitor cells and immune impairment

• 4. Concluding remarks and prospects

• Reviewers

• Acknowledgment

• References

• Biography

• Copyright

1. Introduction

Bone marrow failure syndromes consist of a series of hematological diseases, including acquired aplastic anemia (AA), Fanconi anemia, Diamond-Blackfan syndrome, Shwachfan-Diamond syndrome and Dyskeratosis Congenita [1]. Acquired AA is considered as an immune-mediated disease, which differs from the others associated with genetic pathomechanism. Although acquired AA has been causally associated with many agents including virus, drug, benzene and radiation, no aetiological agent has been identified in most cases. Diverse factors could be held theoretically responsible for bone marrow failure, ranging from immune abnormality, quantitative and qualitative defects in stem/progenitor cells and blocks in differentiation to a lack of stroma support or inadequate cytokine production [2]. Most of AA patients can now be ameliorated or cured by stem-cell transplantation and immunosuppressive therapy, which might be the best evidence of immunologic pathogenesis of acquired AA. Abnormal immunity involved with immune cells and molecules mediated the destruction of stem/progenitor cells with bone marrow hypoplasia instead of fatty or “empty” bone marrow. Thus, more beneficial therapies have been expected to put into practice to acquired AA based on definite immune mechanisms. Therefore, this review will skip, historically and chiastically, the immunologic pathogenesis in order to show immune tissues, cells and molecules’ abnormality and destruction of stem/progenitor cells in acquired AA.

2. Immunologic mechanisms

2.1. Immune tissues

Many scientists and doctors defined acquired AA as a specific marrow failure syndrome. However, lots of evidences display that patients with acquired AA also suffered from other tissues disorder such as thymus, spleen and lymph nodes besides bone marrow. The structure and function of these tissues were damaged following the development of acquired AA.

2.1.1. Bone marrow

The marrow, located in the medullary cavity of bone, is considered as the major postnatal hematopoietic tissue and central immune organ. It consists of hematopoietic stem/progenitor cells (HSCs/HPCs), mesenchymal stem cells (MSCs), angioblasts/endothelial progenitor cells (EPCs) and terminal cells including a variety of stromal cells, hematopoietic cells and immune cells. Bone marrow is severely damaged during the development of acquired AA. Reduced bone marrow capacity, excessive adipocytes, abnormal sinusoidal circulation, reduced blood capillary, interstitial edema and atrophic stroma were observed in animal models [3], [4], [5]. Hematopoietic cells were replaced by a great deal of adipocytes and more lymphocytes, plasma cells and mast cells. As a result, the hematopoietic capacity is diminished significantly to bone marrow hypoplasia and peripheral blood pancytopenia. Based on this feature, magnetic resonance imaging (MRI) may provide an overall estimation of marrow aplasia and identify hypoplastic myelodysplastic syndrome from AA [6], [7].

2.1.2. Thymus, spleen, lymph node

Although acquired AA is defined as a specific marrow failure syndrome, other immune tissues are also damaged. In most cases, hematologists do not find splenectasis and lymphadenectasis during the physical examination except in patients with acquired AA associated with hepatitis. Actually, it hints the damage of spleen and lymph node. To date, there was no report about the pathology of thymus, spleen and lymph node in patients with acquired AA except animal models [3], [5]. Pathological observations showed that spleen, thymus and lymph nodes were remarkably atrophied without extramedullar hematopoiesis, following the bone marrow failure. It appears that drugs, radioactive materials and immunologic mechanism damage thymus, spleen and lymph node together with bone marrow in acquired AA models.

2.2. Immune cells

Patients with acquired AA suffer from varying degree of pancytopenia during the development of disease. The total leukocyte count is low; the hemogram examination reveals a marked decrease in neutrophils, eosinophils and natural killer (NK) cells together with normal or mild lymphopenia. The imbalance of different immune cells count and function reflect abnormal immunity in acquired AA. Reduced and aberrant immune cells weaken the immune response to microorganisms and cause infections readily. Meanwhile, aberrant immunity aggravated the destruction of HSCs/HPCs, MSCs and angioblasts/EPCs.

2.2.1. Innate immune cells

Granulocytes, monocytes, NK cells, NKT cells, dendritic cells (DCs), γδ T cells and B1 cells contribute to innate immunity. They also play important roles in triggering acquired immunity and secreting a variety of cytokines. The majority of previous studies have focused on T and B cells in acquired AA, too little is known about innate immune cells. But, some available data provide strong indication that innate immune cells are abnormal, too.

Obviously, the risks of infections are inversely related to the severity of the reduction of innate immune cells. Decreased neutrophils, monocytes, NK cells and NKT cells in patients with acquired AA resulted in greater susceptibility to infections, even increased the mortality [8], [9], [10], [11], [12], [13], [14], [15]. Defect of phagocytosis was associated with granulocytopenia, monocytopenia and impairment of macrophage-dependent reactions [10]. Eosinophils and basophils appear normal in acquired AA [8]. Both NK cells number and activity was markedly decreased in most patients, and the circulating cells were of the pre-NK cell stage [11], [12], [13]. In most patients treated with antithymocyte globulin (ATG), NK cells returned to the normal range, and recovery of NK cells was correlated to hematopoietic recovery [11]. Similarly, NKT cells were disproportionally decreased in AA marrow, and their deficiency may play a role in the local immune dysregulation [14]. Polarization of NKT cells towards the NKT2 subpopulation occurred after co-stimulation with alpha-galactosylceramide and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in acquired AA [15]. Monocytes, NK cells and NKT cells have been known to play key roles in immune regulation through interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production. But, there have not been rigorous evidence to confirm or exclude whether decreased monocytes, NK cells and NKT cells enhanced IFN-γ and TNF-α impairment of stem/progenitor cells in acquired AA. These results indicate that the defection of monocytes, NK cells and NKT cells is a consequence of the underlying bone marrow failure but not the executant of hematopoietic suppression in acquired AA. However, J.P. Maciejewski et al. [16] showed that percentage of NK cells was elevated in the bone marrow of AA patients but no difference in peripheral blood. During hematopoiesis recovery, NK cells declined, but not to normal levels. This inverse result might support overproduced IFN-γ and IL-2, but is necessary to be investigated and confirmed.

DCs, unlike the above innate immune cells, showed significant difference in acquired AA. The percentage of DCs was elevated in AA patients and associated with the stage of AA [17], [18], [19]. Increased co-stimulatory molecules CD80 and CD86 on DCs might contribute to activate T lymphocyte abnormally [17]. Both immature DC1 and activated DC1 were elevated in the bone marrow of acquired severe aplastic anemia (SAA) patients, and activated DC1 was positively correlated with Th1 and CD8+ T cells but negatively correlated with reticulocytes and neutrophils. DC1 subsets shifted from stable form to active one, which might promote Th0 cells polarization to Th1 cells and trigger the destruction of hematopoiesis [18]. However, the percentage of DC2 was lower than that of controls [19]. After immunesuppresive therapy, immature DC1 and activated DC1 were decreased while DC2 was increased to normal extent [17], [18], [19].

Until now, there is merely a report about γδ T cells but no one about B1 cells in acquired AA. Interestingly, lymphocytes expressing the γδ T-cell receptor (TCR tau delta) were significantly decreased while the proportion of γδ T-cell subpopulation expressing delta TCS1 was markedly increased both before and after 6 weeks of immunosuppressive therapy. Since delta TCS1-positive γδ T cells exhibit auto-immunological capacity, the pathophysiological significance will require further functional analyses [20].

In short, most innate immune cells including neutrophils, monocytes, NK cells and NKT cells were decreased in acquired AA. However, DCs, the professional antigen-presenting cell (APC), were increased and associated with the stage of acquired AA. It is puzzling that decreased monocyte which is another professional APC, undetermined NK cells and γδ T cells co-exist in acquired AA. DCs appear to play a key role in triggering the immune-mediated destruction of hematopoiesis. Defective monocytes, NK cells and NK T cells may be a consequence of the underlying bone marrow failure but not the executant of mediating hematopoietic suppression.

2.2.2. Adaptive immune cells

Acquired immunity depends on B and T lymphocytes. B lymphocytes play important roles in humoral immunity and in activating T lymphocytes. T lymphocytes mediate cellular immunity including enhancing phagocytosis and attacking pathogens. Abnormal acquired immunity occurs together with myeloid deficiency in acquired AA. Although absolute lymphocyte count was normal or decreased in most cases, the relative ratio of lymphocytes was higher than controls [20], [21], [22], [23], [24]. Furthermore, decreased numbers of total lymphocytes, imbalanced T and B subsets have been described in most patients with acquired AA and were associated with impaired bone marrow production of these cells [22], [23], [24]. Interestingly, Fernando Callera et al. demonstrated that Fas-based mediated apoptosis without the apparent participation of bcl-2 or p53 might be a possible mechanism of lymphocyte depletion in patients with AA [25]. Immunosuppressive therapy, bone marrow transplantation (BMT) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) can reverse the phenotypic and functional activity of lymphocytes [20], [22], [26].

Although acquired AA is considered as a disease mediated by T-cell suppression but not B cells which partly failed to produce immunoglobulin and did not response to ATG therapy [12], [24], [27], some available data indicated that autoantibodies were frequently detected in patients with acquired AA [28], [29], [30], [31]. Kinectin, a widely expressed protein, bound to antibodies from 39% of AA patients [28]. Another antigen in a smaller minority of marrow failure patients was diazepam-binding related protein-1 [29]. In addition, the frequent presence of anti-moesin antibody was significantly correlated with the presence of antidiazepam-binding inhibitor-related protein-1 antibody in patients with AA [30]. Anti-moesin antibodies from acquired AA stimulated peripheral blood mononuclear cells (PBMNC) to secrete TNF-α and IFN-γ [31]. Therefore, it cannot exclude whether T-cell and B-cell response to autoantibodies co-exist in acquired AA. It is possible that detection of autoantibodies may be useful in diagnosing and elucidating immune pathophysiology of acquired AA.

T cells have been confirmed to play a major role in the pathogenesis of acquired AA. Cellular and molecular pathways have been mapped in some detail for both effector (T lymphocytes) and target cells (stem/progenitor cells) in the bone marrow. The phenotype, quantity and activity of T lymphocytes are abnormal in many patients with acquired AA. Significantly elevated T cells were found in the areas with residual hematopoiesis, which supported the hypothesis of a site-directed infiltration and local proliferation of T cells in the BM of patients with AA [32]. The helper/inducer: supperssor/cytotoxic T lymphocyte ratio was significantly increased in acquired AA. It showed a return to normal of the T4/T8 in half patients ratio after hematopoietic recovery following ATG treatment [33]. Antigen-driven T-cell responses resulted in oligoclonal T-cell outgrowth, and were involved in the pathogenesis of acquired AA characterized by cyclosporine-dependent recovery of hematopoiesis [34], [35], [36]. Immortalized clones of CD4+ and CD8+ T cells secreted IFN-γ and TNF-α to mediate the destruction of hematopoiesis [37], [38], and were directly toxic to autologous CD34+ cells [34]. By microarray analysis [39], 583 genes, among more than 22,200 transcripts, were differentially expressed in CD3+ T cells derived from the bone marrow of AA patients compared to healthy controls. Dysregulated genes were involved in T-cell mediated cytotoxicity, immune response of Th1 cells, and major regulators of immune function. In hematological remission, the expression levels of candidate genes tended to normalize, such as immune regulators and genes involved in pro-inflammatory immune response. Most of the genes were aberrant in both CD4+ and CD8+ T cells, and consistent with Th1 and Tc1 cell activation. Between CD4+ and CD8+ cells in acquired AA, different genes showed altered expression, implying that their behavior was differently regulated [40].

Th1 lymphocyte is more predominant in the pathogenesis of acquired AA, and it may be very useful to monitor the Th1/Th2 ratio during immunosuppressive therapy [41]. Untreated or refractory patients had a significantly higher proportion of unstimulated Th1 cells that produced IFN-γ and IL-2 whereas Th2 cells did not differ from that of controls, resulting in a shift of IFN-γ/IL-4 ratio towards a type-1 response. Patients in remission also had increased proportion of Th1 cells, with a parallel rise of Th2 cells and normal IFN-γ/IL-4 ratio. So, we presume that DCs might initiate CD4+ cells polarization towards a type-1 response, which in turn leads to activation of cytotoxic CD8+ cells and finally to the destruction of hematopoietic stem cells (HSCs) in newly diagnosed and refractory patients with acquired AA. The polarization of Th1 and increased IFN-γ levels was significantly related with the up-regulated Th1 transcription factor T-bet (T-box expressed in T cells) and T-cell immunoglobulin mucin-3 (TIM-3) [42], [43]. The presence of CD4+ T-cell clones could recognize autologous hematopoietic progenitor cells and increased IFN-γ inhibited the hematopoiesis in acquired AA [44]. Activated CD8+ T cells destroyed hematopoiesis via Fas/FasL induced apoptosis following increased IFN-γ and TNF-α, and cytotoxic mechanism following perforin gene PRF1 mutations [45], [46].

Regulatory T cells, a subpopulation of T cells including CD4+CD25+FOXP3+ Treg, Tr1 and Th3 cells, contribute to keep the balance of immune function. However, CD4+CD25+FOXP3+ Treg cells were decreased in most patients with acquired AA. Meanwhile, all patients examined had low levels of NFAT1 which could explain decreased FOXP3 expression and Treg frequency in acquired AA [47]. Tr1 and Th3 cells in acquired AA remain unclear. Treg defection may diminish the immune regulation of T lymphocytes, and help explain the increased autoreactive T cells during the development of acquired AA.

In general, abnormal immunity in acquired AA not only involves Th1/CD8+ T cells, but also CD4+CD25+ T regulatory cells, B cells, NK cells, DCs, NKT cells, monocytes and neutrophils (Table 1). It shows diverse disorders associated with innate immunity, acquired cellular and humoral immunity in acquired AA. Some alien antigens and autoantigens might induce elevated DCs which promote Th0 cells polarization to Th1 cells, and cause the over-function of Th1 cells and CD8+ T cells. Decreased CD4+CD25+ T regulatory cells, NK cells, NKT cells and monocytes show the deficiency of immune regulation to inhibit excessive Th1 cells and CD8+ cells. As a result, abnormal Th1 and CD8+ T cells destroy predominantly stem/progenitor cells by a variety of immune molecules and signal transducer molecules in acquired AA patients.

Table 1.

Abnormal immune cells in acquired aplastic anemia.


Immune cells	PB	BM	After treatment	References
Granulocytes	Decreased	Decreased	Increased	[8], [9]
Monocytes	Decreased	Decreased	Not done	[8], [9], [10]
NK cells	Decreased	Decreased	Increased	[11], [12], [13]
NK T cells	Decreased	Decreased	Not done	[14], [15]
DCs	Increased	Increased	Decreased	[17], [18], [19]
γδ T cells	Undetermined	Undetermined	Undetermined	[20]
B1 cells	Not done	Not done	Not done
B cells	Decreased	Decreased	Undetermined	[12], [24], [27]

CD4+ T cells
Th1 cells	Increased	Increased	Decreased	[41]
Th2 cells	Normal	Normal	Increased	[41]
CD4+CD25+ Tr	Decreased	Not done	Not done	[47]

CD8+ T cells	Increased	Increased	Decreased	[16], [45]

2.3. Immune molecules

A variety of cytokines are involved in the pathogenesis of acquired AA in autocrine, paracrine and endocrine manners at the hormonal, cellular, and molecular levels. IFN-γ and TNF-α have been confirmed as the key mediators of hematopoietic suppression. In addition, other cytokines, such as interleukins, chemokines and transforming growth factor (TGF) may play important roles during the development of acquired AA. Abnormal cytokines cascade occurs in patients with acquired AA.

Increased production of both IFN-γ and TNF-α have been confirmed to contribute to the pathogenesis of acquired AA, and immunosuppressive therapy may induce hematological remission by suppressing IFN-γ and TNF-α. Previous findings showed excessive production of IFN-γ in most AA patients [37], [38], [41], [48], [49], [50]. And determination of marrow IFN-γ expression was more specific and sensitive than concurrent determinations in peripheral blood [38], [41]. Although IFN-γ can be produced by monocytes, NK cells, NKT cells and B cells as well T cells, it strongly hints that increased DCs, Th1 and CD8+ T cells may be consistent with elevated IFN-γ in acquired AA. T cells have been implicated in the pathogenesis of AA via producing elevated IFN-γ [41], [49], [50], [51]. Circulating IFN-γ containing T cells responded to immunosuppressive therapy and declined after immunosuppressive treatment [37], [49]. The presence of intracellular IFN-γ in T cells may predict the response to immunosuppressive treatment and the onset of AA relapse. Increased IFN-γ can stimulate Th0 cells polarization to Th1 cells and activate CTL cells while antagonizing Th2 cells functions. Thereafter, it also induced secretion of TNF-α and up-regulated Fas and TRAIL expression to induce apoptosis of CD34+ cells together with TNF-α in acquired AA [52], [53], [54]. This was also accompanied by decreased caspase 3 not Bcl-2 family members [54]. p38 MAPK signaling cascade also contributed to the destruction of IFN-γ and TNF-α in human hemopoietic progenitor cells in AA [55]. Inhibition of p38 MAPK reversed the suppressive effects of IFN-γ and TNF-α on normal human bone marrow-derived erythroid and myeloid progenitors. Most importantly, inhibition of p38 MAPK strongly enhanced hematopoietic progenitor colony formation from AA in vitro.

TNF-α is another important cytokine, which was elevated and exhibit synergistic effects with IFN-γ on the suppression of hematopoiesis in patients with acquired AA [50], [56], [57], [58]. Further studies demonstrated that BM lymphocytes, particularly CD4+ and CD8+ T cells, produced significantly higher amounts of TNF-α in patients with AA [51], [59]. Expression of TNF receptor 1 (TNFR1) and TNFR2 in the CD34+ CD38− and CD34+ CD38+ fractions of patients with AA was significantly higher than those in normal control [60]. These results indicate that BM stem cells in patients with AA may be more sensitive to TNF-α. The TNF-a genotype, TNF2 (TNF-308 A) gene, disclosed an immunogenetic association with the pathogenesis and therapeutic response of AA [61]. Similar with IFN-γ, the mechanism of TNF-α suppressing the hematopoiesis was also involved in Fas/FasL, TRAIL and p38 MAPK signaling pathway [54], [55]. Expression of the two cytokines in T cells and their levels in BM plasma showed a decline after therapy compared to pre-therapy of ATG and cyclosporine A [50].

Interleukins play important roles in immune regulation and contribution to hematopoiesis. Lots of interleukins have been evaluated in acquired AA. Interestingly, IL-1, 3, 11 were decreased, IL-4, 10 were normal, and the rest including IL-2, 8, 12, 15, 17, 23 were increased in acquired AA [41], [57], [58], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74]. However, IL-6 acted as a puzziling cytokine with reduced, normal and elevated levels in different studies [64], [66], [70], [75], [76], [77].

It has been confirmed that IL-1, 3, 6 and 11 respectively affect different phase of hematopoiesis in vitro while regulating immune system. Pro-inflammatory cytokine IL-1 regulates adult BM HSCs/HPCs by enhancing the differentiation and proliferation capacity [78], [79], [80]. IL-3 is a multipotent hematopoietic growth factor (HGF), in combination with other cytokines such as stem-cell factor (SCF), IL-6, IL-1, IL-11, G-CSF, GM-CSF, erythropoietin (EPO) and thrombopoietin (TPO), induces the proliferation of colony forming unit-granulocyte-monocyte (CFU-GM), burst forming unit-erythroid (BFU-E), colony forming unit-megakaryocyte (CFU-MK) and mixed lineage colony-forming cells (CFU-GEMM), and stimulates the proliferation of CD34+ cells [79], [81], [82], [83], [84]. IL-11 is a thrombopoietic cytokine that promotes the growth of megakaryocytic progenitors and HSCs, even multilineage progenitors [85], [86]. In addition, IL-11 suppresses production of TNF-α [87]. IL-1, 3 and IL-11 production were markedly reduced in patients with acquired AA. The degree of reduced IL-1 and IL-3 production correlated well with the severity of the disease and the degree of neutropenia [62], [63], [64], [65], [66]. Decreased IL-1, 3, 11 aggravated the bone marrow failure, and returned to normal after successful immunosuppressive treatment. In clinical trials, IL-3 along with other hematopoietic factors has induced sustained remission in patients with AA [88], [89]. Low-dose IL-11 can raise platelet counts without significant toxicity in patients with AA [90], [91]. But, administration of rhIL-1 and IL-3 alone has been disappointing, showing limited efficacy and moderate toxicity [89], [92], [93], [94]. So, it is likely that decreased IL-1, 3, 11 is not a cause of the disease, but merely a consequence. Certainly, it appears that future application of IL-3 and IL-11 in combination with other cytokines is an attractive way in the prevention of acquired AA. IL-6 can be produced by T cells, fibroblasts, macrophages and stromal cells in response to inflammatory and mitogenic stimuli [95]. Although IL-6 alone fails to exert any significant hematopoietic effects in vitro, it acts in synergy with SCF or IL-3 to induce primitive hematopoietic cells to augment myelopoisesis and enhance megakaryocyte development [79], [95], [96], [97]. IL-6 is a confusing cytokine with reduced, normal and elevated levels in different studies about acquired AA. There was no significant correlation between the production of IL-6 and the response to ALG [77]. rhIL-6 given alone at low doses can precipitate a sudden worsening of pre-existing anemia and thrombocytopenia [98]. But, some patients with acquired AA showed IL-6 gene polymorphisms as well IL-6/-174 single nucleotide polymorphism which were linked to high production of TNF-α and IFN-γ [99], [100].

IL-2, 8, 12, 15, 17, 23, which are increased in acquired AA, are potent pro-inflammatory cytokines produced by activated T cells and DCs. Most of them contribute to activation of T cells but inhibit hematopoiesis. IL-2, IL-8, IL-12 and IL-23 were markedly increased in patients with SAA, and seem to be associated with disease activity. Tac antigen (IL-2 receptor) expression on T cells, which can more properly reflect lymphocyte activation, was also increased [101]. After immunosuppressive therapy of ATG and BMT, there was a significant reduction in both the Tac expression and production of IL-2 [102], [103]. Moreover, the efficacy of a monoclonal antibody recognizing IL-2 receptor (daclizumab), has been proven as a successful immunosuppressive agent in AA [104]. Elevated IL-17 can drive the production of TNF-α, IL-6 and IL-8 by a variety of cells including macrophages in AA [78]. IFN-γ can significantly up-regulate IL-15 expression on the surface of AA-BMFSCs (bone marrow fibroblast-like stromal cells), which had the capability to stimulate the proliferation of T lymphocytes. Elevated IL-15 expression can be inhibited by cyclosporin A. Therefore, AA-BMFSCs may indirectly participate in the T-cell-mediated destruction of hematopoietic progenitors by recruiting T cells to BM and stimulating them [74].

IL-4 and IL-10 promote the growth of lymphocytes, especially Th2 and B cells development and activation, and maintain the balance of adaptive immunity. Although IL-4 and IL-10 producing T cells in patients with AA did not differ from that of controls, patients in remission had a parallel rise of IL-4 and IL-10 producing cells along with IFN-γ [41], [75]. It is possible that Th1 cells response is compensated by the increase of IL-4 and IL-10 production [41]. Furthermore, some patients with acquired AA have IL-10/-1082 single nucleotide polymorphism which are linked to high production of TNF-α and IFN-γ [99], [100]. IL-10 was highly effective in reversing hematopoietic suppression by PBMNC from AA patients. The effect was dose dependent and correlated with decreased TNF-α and IFN-γ production. In addition, IL-10 enhanced the erythroid colony formation of progenitor cells by inhibiting the pathological production of IFN-γ. It may be possible to utilize IL-10 as a new supportive therapeutic tool for some AA patients [105], [106].

Macrophage inflammatory protein-1 alpha (MIP-1α), which is known to be mainly produced by monocytes, activated T cells, can contribute to Th1 cells response. Significant difference between the AA and control groups was apparent for MIP-1α in standard long-term marrow cultures (LTMCs) and BMMNC [70], [107]. After LPS and phytohemagglutinin (PHA) stimulation, the production of MIP-1α was markedly increased in the PBMNC of AA patients. Furthermore, the myelopoietic suppressing effect of AA PBMNC-conditioned medium could be significantly blocked by pretreatment with anti-MIP-1α antibody [62], [63]. Increased MIP-1α might contribute to the destruction of hematopoiesis and pathogenesis of acquired AA. TGF-β is a ubiquitous bifunctional cytokine implicated in the regulation of HSCs and bone marrow stromal cells. The lymphocytes of almost all of the AA patients failed to produce detectable amounts of TGF-β. Stimulation of lymphocytes by PHA was known to increase the production of TGF-β in AA [62], [63], [108]. In addition, stromal layers from bone marrow of AA patients produced significantly lower levels of TGF-β1 [109]. There was a correlation between TGF-β1 and platelet count. As TGF-β1 is important in the regulation of haemopoiesis, dysregulation of this cytokine in combination with abnormal other cytokines may contribute significantly to the pathophysiology of AA by exacerbating primary stem-cell defects [109].

In addition, abnormal immunity may also result in the disbalance of diverse HGFs and reduced sensitivity of progenitor cells to the effects of HGFs in acquired AA. Although some HGFs including SCF [110], IL-1, IL-11 and IL-6 were reduced or normal, other specific cytokines such as EPO, TPO, G-CSF and GM-CSF were elevated in acquired AA [77], [111], [112], [113], [114], [115], [116]. Meanwhile, it also suggested the difference that SCF, IL-1, IL-3 and IL-11 affected HSCs in early stage while specific HGFs (EPO, TPO, G-CSF and GM-CSF) promoted specific progenitors in late stage. Elevated HGFs might be due to the reduction of HGFs receptors and accumulation in acquired AA [117]. In SAA, the use of HGFs to support hematopoiesis was of limited value besides G-CSF [118].

In short, lots of cytokines, produced by lymphocytes, monocytes, DCs, stomal cells and endothelial cells, are involved in the pathogenesis of acquired AA (Table 2). Among the abnormal immune molecules, TNF-α and IFN-γ have been confirmed as the major factors mediating the destruction of hematopoiesis. In addition, increased IL-2, 8, 12, 15, 17, 23 and MIP-1α may contribute to the destruction of hematopoiesis in coordination with TNF-α and IFN-γ. Meanwhile, IL-1, 3, 6, 11, with the bifunctions of contribution to hematopoiesis and immune regulation, show lower levels and defect function in AA patients. As a result, both immune destruction and dysfunctional response to a wide range of cytokines stimuli synergistically exacerbate stem/progenitor cells defect in acquired AA.

Table 2.

Abnormal immune molecules in acquired aplastic anemia.


Cytokines	Receptor	PB	BM	Cell sources	After treatment	References
IFN-γ	Not done	Increased	Increased	Th1, CD8+ T cells	Decreased	[37], [38], [41], [48], [49], [50], [51]
TNF-α	Increased	Increased	Increased	CD4+, CD8+ T cells	Decreased	[50], [51], [56], [57], [58], [59], [60]
IL-1	Not done	Decreased	Increased	Monocytes, endothelial cells	Increased	[62], [63], [64]
IL-2	Normal	Increased	Increased	Activated T cells, DCs, monocytes	Decreased	[68], [69]
IL-3	Not done	Decreased	Decreased	PBMNC, stromal cells	Increased	[57], [58], [65], [66]
IL-4	Not done	Normal	Normal	T cells	Increased	[41]
IL-6	Not done	Increased	Increased	BMNC, PBMNC	Decreased	[64], [66], [71], [76], [77], [78]
IL-8	Not done	Increased	Increased	BMNC, PBMNC	Decreased	[70], [71]
IL-10	Not done	Normal	Normal	T cells	Increased	[41], [68]
IL-11	Not done	Decreased	Decreased	PBMNC, stromal cells	Increased	[67]
IL-12	Normal	Increased	Increased	BMNC, PBMNC	Decreased	[72]
IL-15	Not done	Increased	Increased	Stromal cells	Decreased	[73], [74]
IL-17	Not done	Increased	Increased	BMNC, PBMNC	Decreased	[70]
IL-23	Normal	Increased	Increased	BMNC, PBMNC	Decreased	[72]
MIP-1α	Not done	Increased	Increased	PBMNC	Decreased	[62], [63], [70], [107]
TGF-β	Not done	Decreased	Decreased	Stromal cells, lymphocytes	Increased	[62], [63], [108], [109]

Abbreviations: PBMNC, peripheral blood mononuclear cells; BMMNC, bone marrow mononuclear cells; DCs, dendritic cells.

3. Stem/progenitor cells and immune impairment

Pluripotential stem cells derived from totipotential stem cells can differentiate into HSCs, MSCs and angioblasts in the bone marrow. They respectively differentiate into progenitor cells, precursor cells and develop to corresponding terminal cells. HSCs maintain hematopoiesis though the high capacity of proliferation and differentiation. MSCs provide the basis for the physical structures of the microenvironment for HSCs and communicate with hematopoietic cells in different junctions and secreting cytokines to support hematopoiesis during the differentiation into stromal cells. Recent studies demonstrated that bone marrow stromal cells compose two types of niche for HSCs, namely the osteoblastic niche and the vascular niche, in homeostatic regulation of HSC behavior [119], [120]. The niche microenvironments keep HSCs in a dynamic balance between self-renewal and differentiation. Any destruction of these stem/progenitor cells might result in bone marrow failure.

3.1. Hematopoietic stem/progenitor cells and immune impairment

Acquired AA is a syndrome of bone marrow failure characterized by bone marrow hypoplasia and peripheral blood pancytopenia which is secondary to destruction of HSCs/HPCs.

Using clonogenic cultures in vitro, previous investigations demonstrated a marked reduction, or total absence of various phenotypically defined subpopulations of CD34+ cells in acquired AA [121], [122], [123], [124], [125], [126], [127], including long-term culture-initiating cells (LTC-IC), cobblestone area-forming cells (CAFC), and all types of HPCs: CFU-GEMM, CFU-GM, BFU-E and CFU-MK [121], [122], [123], [128], [129], [130], [131], [132], [133]. Meanwhile, CD34+ cells from patients with AA also exhibited qualitative defects [121], [122], [123], [124], [125], [126], [127], [128]. They showed reduced clonogenic potential which was corrected in vitro by the addition of G-CSF. AA BM gave rise to significant lower numbers of committed progenitor cells than normal in clonogenic culture and long-term bone marrow culture (LTBMC) [131], [132], [133]. Furthermore, isolated AA CD34+ cells were dysfunctional in their clonogenic response to a wide range of cytokine stimuli [127]. And decreased HGFs including SCF, IL-1, IL-3 and IL-11 aggravated the deficiency of hematopoiesis in early stage. Although EPO, TPO, G-CSF and GM-CSF were elevated, the reduction of their receptors probably resulted in dysfunctional proliferation and differentiation of HSCs/HPCs. In addition, significantly induced apoptosis of HSCs/HPCs was observed in acquired AA and further aggravated the reduction of HSCs/HPCs [134], [135], [136], [137]. Abnormal immune molecules, especially IFN-γ and TNF-α, induced apoptosis and suppressed the hematopoiesis through Fas/FasL, TRAIL and p38 MAPK signaling pathway [53], [54], [55]. During cellular culture in vitro, extraneous IFN-γ, TNF-α and T cells suppressed hematopoietic colony formation, proliferation and differentiation capacity of normal HSCs/HPCs. As shown previously, increased IL-2, 8, 12, 15, 17, 23 and MIP-1α may contribute to the destruction of hematopoiesis in coordination with TNF-α and IFN-γ. Abnormal immune cells and molecules resulted in a large number of functionally important genes were differentially expressed in HSCs/HPCs.

Weihua Zeng et al. analyzed gene expression profiling and identified a large number of genes expressed differentially in CD34+ cells between AA patients and healthy volunteers [14], [138]. Most genes differentially were up-regulated, which belonged mainly to the functional categories of defense/immune response, cell death and apoptosis, cell cycle/proliferation, cytokine/chemokine, signal transducer, transcription factor and cell adhesion. The genes down-regulated were grouped into cell cycle/proliferation, growth factor, cell growth and maintenance, anti-apoptosis, cell adhesion, oncogenes, signal transduction and immune response. The most striking genes were related to immunity and cell death. A large number of immune/defense response genes were highly expressed in CD34+ cells from AA patients. Almost all of them were up-regulated, including cytokines and cytokine receptors, chemokines and chemokine receptors, signal transduction-mediation genes, and other immune response genes. In addition, apoptosis genes also were differentially expressed in AA. Sixty-seven out of 356 apoptosis genes, including death receptor pathway genes, caspase-related genes, granzyme and perforin pathway genes and other signal transduction-related pathway genes were up-regulated, while three anti-apoptotic regulation genes were down-regulated in AA. Furthermore, cell cycle and cell proliferation genes including 11 signal transduction-related genes, 17 cell proliferation-negative control genes and 6 other cell cycle-related genes were also up-regulated in AA patients. Of these genes, most are believed to exert negative effects on cell proliferation and to inhibit entry into cell cycle. Other researches also confirmed that some of the above genes were differentially increased in acquired AA, including Fas antigen, Bcl-2 and Bcl-x, TNFR2, P-glycoprotein (P-gp), adhesion molecules CD49d and CD49e, and IL-8 [138], [139], [140], [141], [142], while others were decreased such as GATA-2, c-myc and FLT3 [138], [143].

Short telomere has been recently considered as another peculiar feature in acquired AA [144], [145]. Accelerated telomere shortening easily investigated in congenital bone marrow failure has been also identified in about one third of patients with acquired AA [144]. The maintenance of HSCs telomeres is mediated by telomerase [146]. Mutations in telomerase can cause low telomerase activity and further accelerate telomere shortening. HSCs exhaustion is probably secondary to telomere shortening. Systematic surveys disclosed TERC, TERT, TERF1 and TERF2 mutations which was consistent with immune destruction of HSCs [147], [148], [149], [150]. As a result, acquired AA patients with telomerase mutation have low telomerase activity, short telomeres, a hypoplastic bone marrow and reduced hematopoietic function.

Despite the deficit of HSCs/HPCs in acquired AA, autologous hematopoietic reconstitution frequently occurred following immunosuppressive therapy. It hints that a residuary primitive cell population must be normally present in patients with acquired AA. The residuary primitive cells are a promising target population for hematopoietic reconstitution.

3.2. Mesenchymal stem cells and immune impairment

It has been concluded that the defects of HSCs/HPCs, abnormalities of bone marrow microenvironment and immune system disorders are concomitant in acquired AA. When to talk about the replacement of hematopoietically active marrow by fat cells in AA, it seems to be sure that apparent fatty marrow infiltration has been considered a secondary phenomenon. However, Islam A asked a puzziling question that “do bone marrow fat cells or their precursors have a pathogenic role in idiopathic AA” in 1988 [151]. He also postulated that aplastic anemia might result from an abnormal and excessive proliferation of marrow fat cells and the displacement of the hematopoietic tissue of the marrow; and that the resultant marrow failure could be a secondary phenomenon. Then, some researchers began to pay a little more attention to MSCs and the differentiation capacity in acquired AA.

Now, it is generally appreciated that bone marrow microenvironment consists of adipocytes, fibroblasts, osteoblasts, osteoclasts and endothelial cells derived from MSCs. MSCs support hematopoiesis and regulate almost overall immune cells function [152], [153], [154]. Abnormal MSCs, or niche, surely affect the hematopoiesis.

Previous studies showed that there were no significant differences in the expression of MSC markers between in acquired AA and health controls. But, MSCs from patients with AA had poor proliferation potential and deficient support of hematopoietic colony-forming activity [155]. Recent studies also showed the differentiation capacity of MSCs was aberrant in acquired AA. MSCs were more readily induced to differentiate into adipocytes but less readily and slower into osteoblasts, which might correlate with the down-regulation of GATA-2 and overexpression of adipogenic gene-PPARγ in MSCs from patients with acquired AA [156], [157], [158]. MSCs from the bone marrow of SAA patients also showed the immunosuppressive deficiency of T cells activation/proliferation. MSCs were deficient in suppressing MLR, MLR- and PHA-induced T-cell activation/proliferation and IFN-γ release. This functional impairment of MSCs was present in SAA patients at diagnosis, relapse and refractory to immunosuppressive therapy; and recover in remission after treatment. Alternatively, the deficiency of MSCs could be initially induced by autoreactive T cells, and modified MSCs would then be unable to counteract the T-cell activation and IFN-γ production [159]. An animal model study showed that pathogenic T cells from marrow failure donors can destroy HSCs and stromal cells from fully compatible donors, as innocent bystanders: the bystander marrow destructive effect was at least partly mediated by IFN-γ [160]. Bone marrow stromal cells defect could be corrected after BMT [161], [162]. Certainly, it is also hard to exclude that a residuary primitive cell population is present in the bone marrow of acquired AA.

Actually, MSCs in the bone marrow of acquired AA show the defection of hematopoietic stem-cell niche including osteoblastic and vascular niche. Favorable niche not only provides a shelter to protect the stem cell from other stress or challenges but also restrains it from differentiation. Aberrant niche of acquired AA have the poor potential to support hematopoiesis and to maintain the homeostasis of bone marrow. The damaged niche may also be hard to maintain a quiescent state and an immune balance because of immunosuppressive deficiency of over-activated T cells and limited promotion to Treg cells. As a result, abnormal immunity destroys HSCs/HPCs functions to develop to hypoplasia and pancytopenia.

3.3. Angioblasts/endothelial progenitor cells and immune impairment

Recently, angiogenesis was also found to be defective in acquired AA. However, there has been no definite evidence for angioblasts from the bone marrow of patients with acquired AA. Angioblasts can derive from hemangioblasts and mesenchymal stem cells [163], [164]. HSCs and MSCs are deficient in proliferation and differentiation in acquired AA. So we presume that other stem cells as well as angioblasts and hemangioblasts are also damaged in acquired AA. Microvessel density (MVD) can indirectly show the extent of angiogenesis and vascular endothelial growth factor (VEGF) mainly regulates angiogenesis. MVD, serum VEGF levels and VEGF expression were founded to be significantly lower in AA compared to healthy controls. In response to successful immunosupressive therapy and stem-cell transplantation, MVD, serum VEGF levels and VEGF expression in AA increased [165], [166]. Thus, these limited data hint that the vascular niche for HSCs in acquired AA might be defective to form vessels and further weaken the support for hematopoiesis. Likewise, the microvasculature has been described as a critical target in various disorders suffering from enhanced BM angiogenesis such as hematopoietic neoplasms [167], [168]. Therefore, it is very necessary to clarify the vascular niche features in the bone marrow of acquired AA and probably provide sufficient evidence of new therapy.

In view of abnormal immunity and stem/progenitor cells, reasonable and effective immune-regulation and stem/progenitor cells maintenance might be the two therapy targets for acquired AA. Effective immune-regulation therapy will suppress the adverse effects of over-activated DCs, Th1 and CTL cells and promote the favorable function of Treg cells and NK cells, to keep the immune balance. More important and effective therapy is due to available stem/progenitor cells which will provide nourishing niche and sufficient HSCs to maintain hematopoietic homeostasis in the bone marrow. BMT, HSCs transplantation, MSCs transplantation, HSCs and MSCs co-transplantation has been the effective therapy methods for acquired AA. The latest advance of induced pluripotent stem (iPS) cells will probably become the future stem/progenitor candidate for the treatment of AA. The innovation of reprogramming somatic cells to autologous iPS cells has provided a possible new approach to treat beta-thalassemia, Fanconi anemia and sickle cell anemia [169], [170], [171]. It can make the derivation of patient-specific pluripotent cells possible to treat the corresponding disease and avoid the ethics and GVHD. We have a hypothesis that one type of iPS cells, which possess the differentiation capacity into HSCs, MSCs and angioblasts, might be a great candidate for the treatment of acquired AA. It can provide favorable marrow niche, maintain hematopoiesis and keep an immune balance to gain thorough success in the treatment of acquired AA.

4. Concluding remarks and prospects

In summary, it has been confirmed that HSCs/HPCs, MSCs and angioblasts are severely diminished in acquired AA, evidenced by reduced stem/progenitor cell numbers and deficient function. What are the mechanisms of stem/progenitor cell damaged? Abnormal immunity is considered as the major factor mediating the pathogenesis of AA (Fig. 1). Abnormal immune cells and immune molecules are present in AA patients as described previously. Both increased immature DC1 and activated DC1 might promote Th0 cells polarization to Th1 cells, and promote the over-function of T lymphocytes including Th1 cells and CD8+ T cells. In addition, decreased NK cells, NKT cells, monocytes and Treg cells show the deficiency of immune regulation to inhibit excessive Th1 and CD8+ cells. Furthermore, lots of abnormal immune molecules including elevated IFN-γ, TNF-α, MIP-1α and IL-2, 8, 12, 15, 17, 23, produced by disbalanced immune cells and stromal cells, compose a cytokine network to damage stem/progenitor cells. Meanwhile, deficient MSCs and early HGFs including SCF and IL-1, 3, 11 diminish the capacity of immune regulation and aggravate the impairment of hematopoiesis. As a result, a variety of stem/progenitor cells are impaired significantly to be disabled cells together with rare normal ones in bone marrow of AA patients. Impaired stem/progenitor cells show markedly reduced proliferation/differentiation, colony conforming capacity, and increased apoptosis and dysfunctional response to a wide range of cytokine stimuli. The rest normal ones show us the first light of morning in the future. Some patients suffering from acquired AA can now be cured or ameliorated by stem-cell transplantation or immunosuppressive therapy.

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Fig. 1. Abnormal immune destruction of stem/progenitor cells in acquired aplastic anemia. Alien or auto antigens are presented to T lymphocytes by activated DCs, which trigger the polarization of Th0 to Th1 cells and activation of CD8+ T cells. Meanwhile, decreased CD4+CD25+ T cells, NK cells, NKT cells and monocytes show deficiency of immune regulation to inhibit excessive Th1 cells and CD8+ cells. Disbalanced immune cells secrete a variety of immune molecules which compose a cytokine network. Elevated IFN-γ, TNF-α, MIP-1α and IL-2, 8, 12, 15, 17, 23 mediate the destruction of stem/progenitor cells including hematopoietic stem/progenitor cells, mesenchymal stem cells and angioblasts/endothelial progenitor cells. Furthermore, decreased cytokines including TGF-β, IL-1, 3, 11, SCF and damaged mesenchymal stem cells diminish the capacity of immune regulation and aggravate the impairment of hematopoiesis. As a result, a variety of stem/progenitor cells are impaired significantly due to the destruction of proliferation and differentiation capacity, the induction of apoptosis and even shortening telomere. Impaired stem/progenitor cells develop to hypoplasia, reduced angiogenesis and fatty bone marrow during the pathogenesis of acquired aplastic anemia.

However, it is not sufficient for us to enhance the rest normal stem/progenitor cells in bone marrow of AA patients to reconstitution the hematopoiesis. More effective and convenient therapies based on the definite pathogenesis of acquired AA are expected. The patient-specific pluripotent iPS cells, which can differentiate into HSCs, MSCs and angioblasts and maintain immune homeostasis, may be the novel stem/progenitor candidates for the treatment of acquired AA.

Reviewers

Dr. Andrea Bacigalupo, San Martino Hospital, Division of Hematology, Rosanna Benzi 10, I-16132 Genoa, Italy.

Pr. Jakob Robert Passweg, University Hospital of Geneva, Hematology Division, Department of Internal Medicine, Rue Micheli-du-Crest 24, CH-1211 Geneva 14, Switzerland.

Acknowledgements

This study was supported by 863 projects from Ministry Science & Technology of China (2006AA02A110), National Natural Science Foundation of China (30570357 and 30600238) and Tianjin Municipal Science and Technology Commission (06YFSZSF01300 and 07JCYBJC11200).

References

[1]. [1]Shimamura A. Inherited bone marrow failure syndromes: molecular features. Hematology (Am Soc Hematol Educ Program). 2006;63–71.

[2]. [2]Young NS, Calado RT, Scheinberg P. Current concepts in the pathophysiology and treatment of aplastic anemia. Blood. 2006;108:2509–2519. MEDLINE | CrossRef

[3]. [3]Morley A, Blake J. An animal model of chronic aplastic marrow failure. I. Late marrow failure after busulfan. Blood. 1974;44:49–56. MEDLINE

[4]. [4]Knospe WH, Crosby WH. Aplastic anaemia: a disorder of the bone-marrow sinusoidal microcirculation rather than stem-cell failure?. Lancet. 1971;1:20–22. CrossRef

[5]. [5]Pugsley CA, Forbes IJ, Morley AA. Immunologic abnormalities in an animal model of chronic hypoplastic marrow failure induced by busulfan. Blood. 1978;51:601–610. MEDLINE

[6]. [6]Steiner RM, Mitchell DG, Rao VM, Murphy S, Rifkin MD, Burk DL, et al. Magnetic resonance imaging of bone marrow: diagnostic value in diffuse hematologic disorders. Magn Reson Q. 1990;6:17–34. MEDLINE

[7]. [7]Negendank W, Weissman D, Bey TM, de Planque MM, Karanes C, Smith MR, et al. Evidence for clonal disease by magnetic resonance imaging in patients with hypoplastic marrow disorders. Blood. 1991;78:2872–2879. MEDLINE

[8]. [8]Elghetany MT, Hudnall SD, Gardner FH. Peripheral blood picture in primary hypocellular refractory anemia and idiopathic acquired aplastic anemia: an additional tool for differential diagnosis. Haematologica. 1997;82:21–24. MEDLINE

[9]. [9]Keidan AJ, Tsatalas C, Cohen J, Cousins S, Gordon-Smith EC. Infective complications of aplastic anaemia. Br J Haematol. 1986;63:503–508. MEDLINE | CrossRef

[10]. [10]Twomey JJ, Douglass CC, Sharkey O. The monocytopenia of aplastic anemia. Blood. 1973;41:187–195. MEDLINE

[11]. [11]Gascon P, Zoumbos N, Young N. Analysis of natural killer cells in patients with aplastic anemia. Blood. 1986;67:1349–1355. MEDLINE

[12]. [12]Porwit A, Hast R, Stenke L, Wasserman J, Reizenstein P. Decreased blood natural killer cell activity and immunoglobulin synthesis in vitro in aplastic anemia. Acta Med Scand. 1988;224:391–397. MEDLINE | CrossRef

[13]. [13]Taga T, Kariya Y, Shimada M, Uchida A. Suppression of natural killer cell activity by granulocytes in patients with aplastic anemia: role of granulocyte colony-stimulating factor. Immunol Lett. 1993;39:65–70. MEDLINE | CrossRef

[14]. [14]Zeng W, Maciejewski JP, Chen G, Risitano AM, Kirby M, Kajigaya S, et al. Selective reduction of natural killer T cells in the bone marrow of aplastic anaemia. Br J Haematol. 2002;119:803–809. MEDLINE | CrossRef

[15]. [15]Wang Y, Hu X, Guo C, Zhang Q, Peng J, Zhang J, et al. Polarization of natural killer T cells towards an NKT2 subpopulation occurs after stimulation with alpha-galactosylceramide and rhG-CSF in aplastic anemia. Acta Haematol. 2008;119:178–186. CrossRef

[16]. [16]Maciejewski JP, Hibbs JR, Anderson S, Katevas P, Young NS. Bone marrow and peripheral blood lymphocyte phenotype in patients with bone marrow failure. Exp Hematol. 1994;22:1102–1110.

[17]. [17]Tu MF, Shao ZH, Liu H, He GS, Shi J, Bai J, et al. Study on the peripheral blood dendritic cells subtypes and the expression of co-stimulating molecules on dendritic cells and B cells in severe aplastic anemia patients. Zhonghua Xue Ye Xue Za Zhi. 2006;27:611–615. MEDLINE

[18]. [18]He GS, Shao ZH, He H, Liu H, Fu R, Bai J, et al. Changes of subsets of DC1 in the bone marrow of severe aplastic anemia patients. Zhonghua Xue Ye Xue Za Zhi. 2004;25:649–652. MEDLINE

[19]. [19]Wang J, Shao ZH, Fu R, Ruan EB, Qu W, Liang Y, et al. Study on the dendritic cell subsets in peripheral blood and its relationship with the expressions of T-bet and GATA-3 in lymphocytes in severe aplastic anemia. Zhonghua Xue Ye Xue Za Zhi. 2008;29:733–736.

[20]. [20]Mentzel U, Vogt H, Rossol R, Geissler RG, Maurer A, Ganser A, et al. Analysis of lymphocyte subsets in patients with aplastic anemia before and during immunosuppressive therapy. Ann Hematol. 1993;66:127–129. MEDLINE | CrossRef

[21]. [21]Maciejewski JP, Risitano A, Kook H, Zeng W, Chen G, Young NS. Immune pathophysiology of aplastic anemia. Int J Hematol. 2002;76(Suppl. 1):207–214. CrossRef

[22]. [22]de Planque MM, Brand A, Kluin-Nelemans HC, Eernisse JG, van der Burgh F, Natarajan AT, et al. Haematopoietic and immunologic abnormalities in severe aplastic anaemia patients treated with anti-thymocyte globulin. Br J Haematol. 1989;71:421–430. MEDLINE | CrossRef

[23]. [23]Falcao RP, Voltarelli JC, Bottura C. Some immunological studies in aplastic anaemia. J Clin Lab Immunol. 1983;10:25–28. MEDLINE

[24]. [24]Sabbe LJ, Haak HL, Te Velde J, Bradley BA, de Bode L, Blom J, et al. Immunological investigations in aplastic anemia patients. Acta Haematol. 1984;71:178–188. MEDLINE | CrossRef

[25]. [25]Callera F, Garcia AB, Falcao RP. Fas-mediated apoptosis with normal expression of bcl-2 and p53 in lymphocytes from aplastic anaemia. Br J Haematol. 1998;100:698–703. MEDLINE | CrossRef

[26]. [26]Faisal M, Cumberland W, Champlin R, Fahey JL. Effect of recombinant human granulocyte-macrophage colony-stimulating factor administration on the lymphocyte subsets of patients with refractory aplastic anemia. Blood. 1990;76:1580–1585. MEDLINE

[27]. [27]Lum LG, Seigneuret MC, Doney KC, Storb R. In vitro immunoglobulin production, proliferation, and cell markers before and after antithymocyte globulin therapy in patients with aplastic anemia. Am J Hematol. 1987;26:1–15. MEDLINE | CrossRef

[28]. [28]Hirano N, Butler MO, Von Bergwelt-Baildon MS, Maecker B, Schultze JL, O’Connor KC, et al. Autoantibodies frequently detected in patients with aplastic anemia. Blood. 2003;102:4567–4575. MEDLINE | CrossRef

[29]. [29]Feng X, Chuhjo T, Sugimori C, Kotani T, Lu X, Takami A, et al. Diazepam-binding inhibitor-related protein 1: a candidate autoantigen in acquired aplastic anemia patients harboring a minor population of paroxysmal nocturnal hemoglobinuria-type cells. Blood. 2004;104:2425–2431. MEDLINE | CrossRef

[30]. [30]Takamatsu H, Feng X, Chuhjo T, Lu X, Sugimori C, Okawa K, et al. Specific antibodies to moesin, a membrane-cytoskeleton linker protein, are frequently detected in patients with acquired aplastic anemia. Blood. 2007;109:2514–2520. MEDLINE | CrossRef

[31]. [31]Takamatsu H, Espinoza JL, Lu X, Qi Z, Okawa K, Nakao S. Anti-moesin antibodies in the serum of patients with aplastic anemia stimulate peripheral blood mononuclear cells to secrete TNF-alpha and IFN-gamma. J Immunol. 2009;182:703–710.

[32]. [32]Melenhorst JJ, van Krieken JH, Dreef E, Landegent JE, Willemze R, Fibbe WE. T cells selectively infiltrate bone marrow areas with residual haemopoiesis of patients with acquired aplastic anaemia. Br J Haematol. 1997;99:517–519. MEDLINE | CrossRef

[33]. [33]Zoumbos NC, Ferris WO, Hsu SM, Goodman S, Griffith P, Sharrow SO, et al. Analysis of lymphocyte subsets in patients with aplastic anaemia. Br J Haematol. 1984;58:95–105. MEDLINE | CrossRef

[34]. [34]Zeng W, Maciejewski JP, Chen G, Young NS. Limited heterogeneity of T cell receptor BV usage in aplastic anemia. J Clin Invest. 2001;108:765–773. MEDLINE | CrossRef

[35]. [35]Zeng W, Nakao S, Takamatsu H, Yachie A, Takami A, Kondo Y, et al. Characterization of T-cell repertoire of the bone marrow in immune-mediated aplastic anemia: evidence for the involvement of antigen-driven T-cell response in cyclosporine-dependent aplastic anemia. Blood. 1999;93:3008–3016. MEDLINE

[36]. [36]Melenhorst JJ, Fibbe WE, Struyk L, van der Elsen PJ, Willemze R, Landegent JE. Analysis of T-cell clonality in bone marrow of patients with acquired aplastic anaemia. Br J Haematol. 1997;96:85–91. MEDLINE | CrossRef

[37]. [37]Nakao S, Yamaguchi M, Shiobara S, Yokoi T, Miyawaki T, Taniguchi T, et al. Interferon-gamma gene expression in unstimulated bone marrow mononuclear cells predicts a good response to cyclosporine therapy in aplastic anemia. Blood. 1992;79:2532–2535. MEDLINE

[38]. [38]Nistico A, Young NS. gamma-Interferon gene expression in the bone marrow of patients with aplastic anemia. Ann Intern Med. 1994;120:463–469. MEDLINE

[39]. [39]Franzke A, Geffers R, Hunger JK, Pfortner S, Piao W, Ivanyi P, et al. Identification of novel regulators in T-cell differentiation of aplastic anemia patients. BMC Genomics. 2006;7:263. MEDLINE | CrossRef

[40]. [40]Zeng W, Kajigaya S, Chen G, Risitano AM, Nunez O, Young NS. Transcript profile of CD4+ and CD8+ T cells from the bone marrow of acquired aplastic anemia patients. Exp Hematol. 2004;32:806–814. CrossRef

[41]. [41]Giannakoulas NC, Karakantza M, Theodorou GL, Pagoni M, Galanopoulos A, Kakagianni T, et al. Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients. Br J Haematol. 2004;124:97–105. MEDLINE | CrossRef

[42]. [42]Solomou EE, Keyvanfar K, Young NS. T-bet, a Th1 transcription factor, is up-regulated in T cells from patients with aplastic anemia. Blood. 2006;107:3983–3991. MEDLINE | CrossRef

[43]. [43]Zhang J, Gu Y, Xu C, Qu X. Increased T cell immunoglobulin mucin-3 and its ligand in acquired aplastic anemia. Eur J Haematol. 2008;81:130–139. CrossRef

[44]. [44]Nakao S, Takamatsu H, Yachie A, Itoh T, Yamaguchi M, Ueda M, et al. Establishment of a CD4+ T cell clone recognizing autologous hematopoietic progenitor cells from a patient with immune-mediated aplastic anemia. Exp Hematol. 1995;23:433–438.

[45]. [45]Zoumbos NC, Gascon P, Djeu JY, Trost SR, Young NS. Circulating activated suppressor T lymphocytes in aplastic anemia. N Engl J Med. 1985;312:257–265. MEDLINE | CrossRef

[46]. [46]Solomou EE, Gibellini F, Stewart B, Malide D, Berg M, Visconte V, et al. Perforin gene mutations in patients with acquired aplastic anemia. Blood. 2007;109:5234–5237. MEDLINE | CrossRef

[47]. [47]Solomou EE, Rezvani K, Mielke S, Malide D, Keyvanfar K, Visconte V, et al. Deficient CD4+ CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia. Blood. 2007;110:1603–1606. CrossRef

[48]. [48]Zoumbos NC, Gascon P, Djeu JY, Young NS. Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo. Proc Natl Acad Sci U S A. 1985;82:188–192. MEDLINE | CrossRef

[49]. [49]Sloand E, Kim S, Maciejewski JP, Tisdale J, Follmann D, Young NS. Intracellular interferon-gamma in circulating and marrow T cells detected by flow cytometry and the response to immunosuppressive therapy in patients with aplastic anemia. Blood. 2002;100:1185–1191. MEDLINE | CrossRef

[50]. [50]Dubey S, Shukla P, Nityanand S. Expression of interferon-gamma and tumor necrosis factor-alpha in bone marrow T cells and their levels in bone marrow plasma in patients with aplastic anemia. Ann Hematol. 2005;84:572–577. MEDLINE | CrossRef

[51]. [51]Dufour C, Corcione A, Svahn J, Haupt R, Battilana N, Pistoia V. Interferon gamma and tumour necrosis factor alpha are overexpressed in bone marrow T lymphocytes from paediatric patients with aplastic anaemia. Br J Haematol. 2001;115:1023–1031. MEDLINE | CrossRef

[52]. [52]Rose DM, Winston BW, Chan ED, Riches DW, Henson PM. Interferon-gamma and transforming growth factor-beta modulate the activation of mitogen-activated protein kinases and tumor necrosis factor-alpha production induced by Fc gamma-receptor stimulation in murine macrophages. Biochem Biophys Res Commun. 1997;238:256–260. CrossRef

[53]. [53]Kakagianni T, Giannakoulas NC, Thanopoulou E, Galani A, Michalopoulou S, Kouraklis-Symeonidis A, et al. A probable role for trail-induced apoptosis in the pathogenesis of marrow failure. Implications from an in vitro model and from marrow of aplastic anemia patients. Leuk Res. 2006;30:713–721. Abstract | Full Text | Full-Text PDF (343 KB) | CrossRef

[54]. [54]Maciejewski J, Selleri C, Anderson S, Young NS. Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro. Blood. 1995;85:3183–3190. MEDLINE

[55]. [55]Verma A, Deb DK, Sassano A, Kambhampati S, Wickrema A, Uddin S, et al. Cutting edge: activation of the p38 mitogen-activated protein kinase signaling pathway mediates cytokine-induced hemopoietic suppression in aplastic anemia. J Immunol. 2002;168:5984–5988. MEDLINE

[56]. [56]Schultz JC, Shahidi NT. Detection of tumor necrosis factor-alpha in bone marrow plasma and peripheral blood plasma from patients with aplastic anemia. Am J Hematol. 1994;45:32–38. MEDLINE | CrossRef

[57]. [57]Hsu HC, Tsai WH, Chen LY, Hsu ML, Ho CH, Lin CK, et al. Overproduction of inhibitory hematopoietic cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells in patients with aplastic anemia. Ann Hematol. 1995;71:281–286. MEDLINE | CrossRef

[58]. [58]Hsu HC, Tsai WH, Chen LY, Hsu ML, Ing-Tiau Kuo B, Ho CH, et al. Production of hematopoietic regulatory cytokines by peripheral blood mononuclear cells in patients with aplastic anemia. Exp Hematol. 1996;24:31–36.

[59]. [59]Hara T, Ando K, Tsurumi H, Moriwaki H. Excessive production of tumor necrosis factor-alpha by bone marrow T lymphocytes is essential in causing bone marrow failure in patients with aplastic anemia. Eur J Haematol. 2004;73:10–16. MEDLINE | CrossRef

[60]. [60]Kasahara S, Hara T, Itoh H, Ando K, Tsurumi H, Sawada M, et al. Hypoplastic myelodysplastic syndromes can be distinguished from acquired aplastic anaemia by bone marrow stem cell expression of the tumour necrosis factor receptor. Br J Haematol. 2002;118:181–188. MEDLINE | CrossRef

[61]. [61]Demeter J, Messer G, Schrezenmeier H. Clinical relevance of the TNF-alpha promoter/enhancer polymorphism in patients with aplastic anemia. Ann Hematol. 2002;81:566–569. MEDLINE | CrossRef

[62]. [62]Gascon P, Scala G. Decreased interleukin-1 production in aplastic anemia. Am J Med. 1988;85:668–674. Abstract | Full-Text PDF (783 KB) | CrossRef

[63]. [63]Nakao S, Matsushima K, Young N. Decreased interleukin 1 production in aplastic anaemia. Br J Haematol. 1989;71:431–436. MEDLINE | CrossRef

[64]. [64]Holmberg LA, Seidel K, Leisenring W, Torok-Storb B. Aplastic anemia: analysis of stromal cell function in long-term marrow cultures. Blood. 1994;84:3685–3690. MEDLINE

[65]. [65]Kawano Y, Takaue Y, Hirao A, Watanabe T, Abe T, Shimizu T, et al. Production of interleukin 3 and granulocyte-macrophage colony-stimulating factor from stimulated blood mononuclear cells in patients with aplastic anemia. Exp Hematol. 1992;20:1125–1128.

[66]. [66]Gibson FM, Scopes J, Daly S, Ball S, Gordon-Smith EC. Haemopoietic growth factor production by normal and aplastic anaemia stroma in long-term bone marrow culture. Br J Haematol. 1995;91:551–561. MEDLINE | CrossRef

[67]. [67]Cockrell EM, Gorman J, Hord JD. Endogenous interleukin-11 (IL-11) levels in newly diagnosed children with acquired severe aplastic anemia (SAA). Cytokine. 2004;28:55–58. MEDLINE | CrossRef

[68]. [68]Gascon P, Zoumbos NC, Scala G, Djeu JY, Moore JG, Young NS. Lymphokine abnormalities in aplastic anemia: implications for the mechanism of action of antithymocyte globulin. Blood. 1985;65:407–413. MEDLINE

[69]. [69]Nissen C, Moser Y, Weis J, Wursch A, Gratwohl A, Speck B. The release of interleukin-2 (IL-2) and colony stimulating activity (CSA) in aplastic anemia patients: opposite behaviour with improvement of bone marrow function. Blut. 1986;52:221–230.

[70]. [70]Gu Y, Hu X, Liu C, Qv X, Xu C. Interleukin (IL)-17 promotes macrophages to produce IL-8, IL-6 and tumour necrosis factor-alpha in aplastic anaemia. Br J Haematol. 2008;142:109–114. CrossRef

[71]. [71]Tripathy NK, Nityanand S. Bone marrow and blood plasma levels of IL-8 in aplastic anemia and their relationship with disease severity. Am J Hematol. 2005;79:240–242. MEDLINE | CrossRef

[72]. [72]Gu Y, Zhang J, Peng J, Hu X, Xu C. Elevated expression of IL-12 and IL-23 in patients with aplastic anemia. Int J Lab Hematol. 2009;31:207–214.

[73]. [73]Dirksen U, Asadi-Moghaddam K, Fuhrer M, Burdach S. Defunct hematopoietic progenitor growth and heterogeneous immunological phenotypes in acquired aplastic anemia of childhood: identification of subsets with decreased hematopoietic progenitors and increased IL15 or IL10 production. Klin Padiatr. 1998;210:167–172. MEDLINE | CrossRef

[74]. [74]Wenxin L, Jinxiang F, Yong W, Wenxiang L, Wenbiao S, Xueguang Z. Expression of membrane-bound IL-15 by bone marrow fibroblast-like stromal cells in aplastic anemia. Int Immunol. 2005;17:429–437. MEDLINE | CrossRef

[75]. [75]Dilloo D, Vohringer R, Josting A, Habersang K, Scheidt A, Burdach S. Bone marrow fibroblasts from children with aplastic anemia exhibit reduced interleukin-6 production in response to cytokines and viral challenge. Pediatr Res. 1995;38:716–721. MEDLINE

[76]. [76]Mayer A, Podlech J, Kurz S, Steffens HP, Maiberger S, Thalmeier K, et al. Bone marrow failure by cytomegalovirus is associated with an in vivo deficiency in the expression of essential stromal hemopoietin genes. J Virol. 1997;71:4589–4598.

[77]. [77]Kojima S, Matsuyama T, Kodera Y. Hematopoietic growth factors released by marrow stromal cells from patients with aplastic anemia. Blood. 1992;79:2256–2261. MEDLINE

[78]. [78]Dinarello CA. Biologic basis for interleukin-1 in disease. Blood. 1996;87:2095–2147. MEDLINE

[79]. [79]Brugger W, Mocklin W, Heimfeld S, Berenson RJ, Mertelsmann R, Kanz L. Ex vivo expansion of enriched peripheral blood CD34+ progenitor cells by stem cell factor, interleukin-1 beta (IL-1 beta), IL-6, IL-3, interferon-gamma, and erythropoietin. Blood. 1993;81:2579–2584. MEDLINE

[80]. [80]Jacobsen SE, Ruscetti FW, Okkenhaug C, Lien E, Ortiz M, Veiby OP, et al. Distinct and direct synergistic effects of IL-1 and IL-6 on proliferation and differentiation of primitive murine hematopoietic progenitor cells in vitro. Exp Hematol. 1994;22:1064–1069.

[81]. [81]Farese AM, Williams DE, Seiler FR, MacVittie TJ. Combination protocols of cytokine therapy with interleukin-3 and granulocyte-macrophage colony-stimulating factor in a primate model of radiation-induced marrow aplasia. Blood. 1993;82:3012–3018. MEDLINE

[82]. [82]Lindemann A, Mertelsmann R. Interleukin-3 and its receptor. Cancer Treat Res. 1995;80:107–142. MEDLINE

[83]. [83]Eder M, Geissler G, Ganser A. IL-3 in the clinic. Stem Cells. 1997;15:327–333. MEDLINE | CrossRef

[84]. [84]Metcalf D, Begley CG, Johnson GR, Nicola NA, Lopez AF, Williamson DJ. Effects of purified bacterially synthesized murine multi-CSF (IL-3) on hematopoiesis in normal adult mice. Blood. 1986;68:46–57. MEDLINE

[85]. [85]Kaushansky K, Thrombopoietin . N Engl J Med. 1998;339:746–754. MEDLINE | CrossRef

[86]. [86]Kurzrock R. Thrombopoietic factors in chronic bone marrow failure states: the platelet problem revisited. Clin Cancer Res. 2005;11:1361–1367. MEDLINE | CrossRef

[87]. [87]Cork BA, Tuckerman EM, Li TC, Laird SM. Expression of interleukin (IL)-11 receptor by the human endometrium in vivo and effects of IL-11, IL-6 and LIF on the production of MMP and cytokines by human endometrial cells in vitro. Mol Hum Reprod. 2002;8:841–848. MEDLINE | CrossRef

[88]. [88]Wu HH, Talpaz M, Champlin RE, Pilat SR, Kurzrock R. Sequential interleukin 3 and granulocyte-macrophage-colony stimulating factor therapy in patients with bone marrow failure with long-term follow-up of responses. Cancer. 2003;98:2410–2419.

[89]. [89]Raghavachar A, Ganser A, Freund M, Heimpel H, Herrmann F, Schrezenmeier H. Long-term interleukin-3 and intensive immunosuppression in the treatment of aplastic anemia. Cytokines Mol Ther. 1996;2:215–223. MEDLINE

[90]. [90]Kurzrock R, Cortes J, Thomas DA, Jeha S, Pilat S, Talpaz M. Pilot study of low-dose interleukin-11 in patients with bone marrow failure. J Clin Oncol. 2001;19:4165–4172.

[91]. [91]Tsimberidou AM, Giles FJ, Khouri I, Bueso-Ramos C, Pilat S, Thomas DA, et al. Low-dose interleukin-11 in patients with bone marrow failure: update of the M. D. Anderson Cancer Center experience. Ann Oncol. 2005;16:139–145. MEDLINE | CrossRef

[92]. [92]Walsh CE, Liu JM, Anderson SM, Rossio JL, Nienhuis AW, Young NS. A trial of recombinant human interleukin-1 in patients with severe refractory aplastic anaemia. Br J Haematol. 1992;80:106–110. MEDLINE | CrossRef

[93]. [93]Nemunaitis J, Ross M, Meisenberg B, O’Reilly R, Lilleby K, Buckner CD, et al. Phase I study of recombinant human interleukin-1 beta (rhIL-1 beta) in patients with bone marrow failure. Bone Marrow Transplant. 1994;14:583–588. MEDLINE

[94]. [94]Ganser A, Lindemann A, Seipelt G, Ottmann OG, Eder M, Falk S, et al. Effects of recombinant human interleukin-3 in aplastic anemia. Blood. 1990;76:1287–1292. MEDLINE

[95]. [95]Kishimoto T. The biology of interleukin-6. Blood. 1989;74:1–10. MEDLINE

[96]. [96]Ulich TR, del Castillo J, Guo KZ. In vivo hematologic effects of recombinant interleukin-6 on hematopoiesis and circulating numbers of RBCs and WBCs. Blood. 1989;73:108–110. MEDLINE

[97]. [97]Carrington PA, Hill RJ, Stenberg PE, Levin J, Corash L, Schreurs J, et al. Multiple in vivo effects of interleukin-3 and interleukin-6 on murine megakaryocytopoiesis. Blood. 1991;77:34–41. MEDLINE

[98]. [98]Schrezenmeier H, Marsh JC, Stromeyer P, Muller H, Heimpel H, Gordon-Smith EC, et al. A phase I/II trial of recombinant human interleukin-6 in patients with aplastic anaemia. Br J Haematol. 1995;90:283–292. MEDLINE | CrossRef

[99]. [99]Fermo E, Bianchi P, Barcellini W, Pedotti P, Boschetti C, Alfinito F, et al. Immunoregulatory cytokine polymorphisms in Italian patients affected by paroxysmal nocturnal haemoglobinuria and aplastic anaemia. Eur J Immunogenet. 2004;31:267–269. MEDLINE | CrossRef

[100]. [100]Gidvani V, Ramkissoon S, Sloand EM, Young NS. Cytokine gene polymorphisms in acquired bone marrow failure. Am J Hematol. 2007;82:721–724. CrossRef

[101]. [101]Hinterberger W, Adolf G, Bettelheim P, Geissler K, Huber C, Irschick E, et al. Lymphokine overproduction in severe aplastic anemia is not related to blood transfusions. Blood. 1989;74:2713–2717. MEDLINE

[102]. [102]Azogui O, Gluckman E, Fradelizi D. Inhibiton of IL 2 production after human allogeneic bone marrow transplantation. J Immunol. 1983;131:1205–1208. MEDLINE

[103]. [103]Platanias L, Gascon P, Bielory L, Griffith P, Nienhuis A, Young N. Lymphocyte phenotype and lymphokines following anti-thymocyte globulin therapy in patients with aplastic anaemia. Br J Haematol. 1987;66:437–443. MEDLINE | CrossRef

[104]. [104]Maciejewski JP, Sloand EM, Nunez O, Boss C, Young NS. Recombinant humanized anti-IL-2 receptor antibody (daclizumab) produces responses in patients with moderate aplastic anemia. Blood. 2003;102:3584–3586. MEDLINE | CrossRef

[105]. [105]Geissler K, Kabrna E, Kollars M, Ohler L, Berer A, Burgmann H, et al. Interleukin-10 inhibits in vitro hematopoietic suppression and production of interferon-gamma and tumor necrosis factor-alpha by peripheral blood mononuclear cells from patients with aplastic anemia. Hematol J. 2002;3:206–213. MEDLINE | CrossRef

[106]. [106]Asano Y, Shibata S, Kobayashi S, Okamura S, Niho Y. Effect of interleukin 10 on the hematopoietic progenitor cells from patients with aplastic anemia. Stem Cells. 1999;17:147–151. MEDLINE | CrossRef

[107]. [107]Maciejewski JP, Liu JM, Green SW, Walsh CE, Plumb M, Pragnell IB, et al. Expression of stem cell inhibitor (SCI) gene in patients with bone marrow failure. Exp Hematol. 1992;20:1112–1117.

[108]. [108]Taketazu F, Miyagawa K, Ichijo H, Oshimi K, Mizoguchi H, Hirai H, et al. Decreased level of transforming growth factor-beta in blood lymphocytes of patients with aplastic anemia. Growth Factors. 1992;6:85–90. MEDLINE | CrossRef

[109]. [109]Rizzo S, Killick SB, Patel S, Ball SE, Wadhwa M, Dilger P, et al. Reduced TGF-beta1 in patients with aplastic anaemia in vivo and in vitro. Br J Haematol. 1999;107:797–803. MEDLINE | CrossRef

[110]. [110]Wodnar-Filipowicz A, Yancik S, Moser Y, dalle Carbonare V, Gratwohl A, Tichelli A, et al. Levels of soluble stem cell factor in serum of patients with aplastic anemia. Blood. 1993;81:3259–3264. MEDLINE

[111]. [111]Das RE, Milne A, Rowley M, Smith EC, Cotes PM. Serum immunoreactive erythropoietin in patients with idiopathic aplastic and Fanconi's anaemias. Br J Haematol. 1992;82:601–607. MEDLINE | CrossRef

[112]. [112]Schrezenmeier H, Noe G, Raghavachar A, Rich IN, Heimpel H, Kubanek B. Serum erythropoietin and serum transferrin receptor levels in aplastic anaemia. Br J Haematol. 1994;88:286–294. MEDLINE | CrossRef

[113]. [113]Marsh JC, Gibson FM, Prue RL, Bowen A, Dunn VT, Hornkohl AC, et al. Serum thrombopoietin levels in patients with aplastic anaemia. Br J Haematol. 1996;95:605–610. MEDLINE

[114]. [114]Schrezenmeier H, Griesshammer M, Hornkohl A, Nichol JL, Hecht T, Heimpel H, et al. Thrombopoietin serum levels in patients with aplastic anaemia: correlation with platelet count and persistent elevation in remission. Br J Haematol. 1998;100:571–576. MEDLINE | CrossRef

[115]. [115]Kojima S, Matsuyama T, Kodera Y, Tahara T, Kato T. Measurement of endogenous plasma thrombopoietin in patients with acquired aplastic anaemia by a sensitive enzyme-linked immunosorbent assay. Br J Haematol. 1997;97:538–543. MEDLINE

[116]. [116]Watari K, Asano S, Shirafuji N, Kodo H, Ozawa K, Takaku F, et al. Serum granulocyte colony-stimulating factor levels in healthy volunteers and patients with various disorders as estimated by enzyme immunoassay. Blood. 1989;73:117–122. MEDLINE

[117]. [117]Xu H, Li A, Yu Y, Li J, Liu X, Wang X, et al. Comparative analysis of G-CSFR and GM-CSFR expressions on CD(34)(+) cells in patients with aplastic anemia and myelodysplastic syndrome. Int J Lab Hematol. 2008;.

[118]. [118]Marsh JC, Ganser A, Stadler M. Hematopoietic growth factors in the treatment of acquired bone marrow failure states. Semin Hematol. 2007;44:138–147. Abstract | Full Text | Full-Text PDF (157 KB) | CrossRef

[119]. [119]Li Z, Li L. Understanding hematopoietic stem-cell microenvironments. Trends Biochem Sci. 2006;31:589–595. MEDLINE | CrossRef

[120]. [120]Shiozawa Y, Havens AM, Pienta KJ, Taichman RS. The bone marrow niche: habitat to hematopoietic and mesenchymal stem cells, and unwitting host to molecular parasites. Leukemia. 2008;22:941–950. CrossRef

[121]. [121]Maciejewski JP, Selleri C, Sato T, Anderson S, Young NS. A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia. Blood. 1996;88:1983–1991. MEDLINE

[122]. [122]Podesta M, Piaggio G, Frassoni F, Pitto A, Zikos P, Sessarego M, et al. The assessment of the hematopoietic reservoir after immunosuppressive therapy or bone marrow transplantation in severe aplastic anemia. Blood. 1998;91:1959–1965. MEDLINE

[123]. [123]Rizzo S, Scopes J, Elebute MO, Papadaki HA, Gordon-Smith EC, Gibson FM. Stem cell defect in aplastic anemia: reduced long term culture-initiating cells (LTC-IC) in CD34+ cells isolated from aplastic anemia patient bone marrow. Hematol J. 2002;3:230–236. MEDLINE | CrossRef

[124]. [124]Manz CY, Nissen C, Wodnar-Filipowicz A. Deficiency of CD34+ c-kit+ and CD34+38- hematopoietic precursors in aplastic anemia after immunosuppressive treatment. Am J Hematol. 1996;52:264–274. MEDLINE | CrossRef

[125]. [125]Rizzo S, Scopes J, Draycott GS, Pocock C, Foukaneli T, Rutherford TR, et al. Quiescent (5-fluorouracil-resistant) aplastic anemia hematopoietic cells in vitro. Exp Hematol. 2004;32:665–672. CrossRef

[126]. [126]Matsui WH, Brodsky RA, Smith BD, Borowitz MJ, Jones RJ. Quantitative analysis of bone marrow CD34 cells in aplastic anemia and hypoplastic myelodysplastic syndromes. Leukemia. 2006;20:458–462. MEDLINE | CrossRef

[127]. [127]Scopes J, Daly S, Atkinson R, Ball SE, Gordon-Smith EC, Gibson FM. Aplastic anemia: evidence for dysfunctional bone marrow progenitor cells and the corrective effect of granulocyte colony-stimulating factor in vitro. Blood. 1996;87:3179–3185. MEDLINE

[128]. [128]Schrezenmeier H, Jenal M, Herrmann F, Heimpel H, Raghavachar A. Quantitative analysis of cobblestone area-forming cells in bone marrow of patients with aplastic anemia by limiting dilution assay. Blood. 1996;88:4474–4480. MEDLINE

[129]. [129]Bacigalupo A, Piaggio G, Podesta M, Tong J, Pitto A, Figari O, et al. Early hemopoietic progenitors in the peripheral blood of patients with severe aplastic anemia (SAA) after treatment with antilymphocyte globulin (ALG), cyclosporin-A and G-CSF. Haematologica. 1997;82:133–137. MEDLINE

[130]. [130]Cox CV, Killick SB, Patel S, Elebute MO, Marsh JC, Gordon-Smith EC, et al. In vitro proliferation and differentiation of megakaryocytic progenitors in patients with aplastic anemia, paroxysmal nocturnal hemoglobinuria, and the myelodysplastic syndromes. Stem Cells (Dayton, Ohio). 2000;18:428–434. MEDLINE | CrossRef

[131]. [131]Marsh JC, Chang J, Testa NG, Hows JM, Dexter TM. The hematopoietic defect in aplastic anemia assessed by long-term marrow culture. Blood. 1990;76:1748–1757. MEDLINE

[132]. [132]Bacigalupo A, Figari O, Tong J, Piaggio G, Miceli S, Frassoni F, et al. Long-term marrow culture in patients with aplastic anemia compared with marrow transplant recipients and normal controls. Exp Hematol. 1992;20:425–430.

[133]. [133]Scopes J, Bagnara M, Gordon-Smith EC, Ball SE, Gibson FM. Haemopoietic progenitor cells are reduced in aplastic anaemia. Br J Haematol. 1994;86:427–430. MEDLINE | CrossRef

[134]. [134]Philpott NJ, Scopes J, Marsh JC, Gordon-Smith EC, Gibson FM. Increased apoptosis in aplastic anemia bone marrow progenitor cells: possible pathophysiologic significance. Exp Hematol. 1995;23:1642–1648.

[135]. [135]Timeus F, Crescenzio N, Doria A, Foglia L, Linari A, Giaccone M, et al. Flow cytometric evaluation of circulating CD34+ cell counts and apoptotic rate in children with acquired aplastic anemia and myelodysplasia. Exp Hematol. 2005;33:597–604. CrossRef

[136]. [136]Callera F, Falcao RP. Increased apoptotic cells in bone marrow biopsies from patients with aplastic anaemia. Br J Haematol. 1997;98:18–20. MEDLINE

[137]. [137]Killick SB, Cox CV, Marsh JC, Gordon-Smith EC, Gibson FM. Mechanisms of bone marrow progenitor cell apoptosis in aplastic anaemia and the effect of anti-thymocyte globulin: examination of the role of the Fas-Fas-L interaction. Br J Haematol. 2000;111:1164–1169. MEDLINE | CrossRef

[138]. [138]Zeng W, Chen G, Kajigaya S, Nunez O, Charrow A, Billings EM, et al. Gene expression profiling in CD34 cells to identify differences between aplastic anemia patients and healthy volunteers. Blood. 2004;103:325–332. MEDLINE | CrossRef

[139]. [139]Maciejewski JP, Selleri C, Sato T, Anderson S, Young NS. Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia. Br J Haematol. 1995;91:245–252. MEDLINE | CrossRef

[140]. [140]Ismail M, Gibson FM, Gordon-Smith EC, Rutherford TR. Bcl-2 and Bcl-x expression in the CD34+ cells of aplastic anaemia patients: relationship with increased apoptosis and upregulation of Fas antigen. Br J Haematol. 2001;113:706–712. MEDLINE | CrossRef

[141]. [141]Calado RT, Garcia AB, Gallo DA, Falcao RP. Reduced function of the multidrug resistance P-glycoprotein in CD34+ cells of patients with aplastic anaemia. Br J Haematol. 2002;118:320–326. MEDLINE | CrossRef

[142]. [142]Kaito K, Otsubo H, Ogasawara Y, Shimada T, Usui N, Kobayashi M. Adhesion molecule expression by bone marrow CD34-positive cells in aplastic anemia before and after immunosuppressive therapy. Clin Lab Haematol. 2003;25:393–396. MEDLINE | CrossRef

[143]. [143]Fujimaki S, Harigae H, Sugawara T, Takasawa N, Sasaki T, Kaku M. Decreased expression of transcription factor GATA-2 in haematopoietic stem cells in patients with aplastic anaemia. Br J Haematol. 2001;113:52–57. MEDLINE | CrossRef

[144]. [144]Ball SE, Gibson FM, Rizzo S, Tooze JA, Marsh JC, Gordon-Smith EC. Progressive telomere shortening in aplastic anemia. Blood. 1998;91:3582–3592. MEDLINE

[145]. [145]Brummendorf TH, Maciejewski JP, Mak J, Young NS, Lansdorp PM. Telomere length in leukocyte subpopulations of patients with aplastic anemia. Blood. 2001;97:895–900. MEDLINE | CrossRef

[146]. [146]Calado RT, Young NS. Telomere maintenance and human bone marrow failure. Blood. 2008;111:4446–4455. CrossRef

[147]. [147]Fogarty PF, Yamaguchi H, Wiestner A, Baerlocher GM, Sloand E, Zeng WS, et al. Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA. Lancet. 2003;362:1628–1630. Abstract | Full Text | Full-Text PDF (68 KB) | CrossRef

[148]. [148]Yamaguchi H. Mutations of telomerase complex genes linked to bone marrow failures. J Nippon Med Sch = Nihon Ika Daigaku zasshi. 2007;74:202–209.

[149]. [149]Yamaguchi H, Calado RT, Ly H, Kajigaya S, Baerlocher GM, Chanock SJ, et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med. 2005;352:1413–1424. CrossRef

[150]. [150]Savage SA, Calado RT, Xin ZT, Ly H, Young NS, Chanock SJ. Genetic variation in telomeric repeat binding factors 1 and 2 in aplastic anemia. Exp Hematol. 2006;34:664–671. CrossRef

[151]. [151]Islam A. Do bone marrow fat cells or their precursors have a pathogenic role in idiopathic aplastic anaemia?. Med Hypotheses. 1988;25:209–217. MEDLINE | CrossRef

[152]. [152]Le Blanc K, Samuelsson H, Gustafsson B, Remberger M, Sundberg B, Arvidson J, et al. Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia. 2007;21:1733–1738. CrossRef

[153]. [153]Fang B, Li N, Song Y, Li J, Zhao RC, Ma Y. Cotransplantation of haploidentical mesenchymal stem cells to enhance engraftment of hematopoietic stem cells and to reduce the risk of graft failure in two children with severe aplastic anemia. Pediatr Transplant. 2008;.

[154]. [154]Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev. 2008;8:726–736.

[155]. [155]Juneja HS, Gardner FH. Functionally abnormal marrow stromal cells in aplastic anemia. Exp Hematol. 1985;13:194–199.

[156]. [156]Xu Y, Takahashi Y, Wang Y, Hama A, Nishio N, Muramatsu H, et al. Downregulation of GATA-2 and overexpression of adipogenic gene-PPARgamma in mesenchymal stem cells from patients with aplastic anemia. Exp Hematol. 2009;37:1393–1399. CrossRef

[157]. [157]Wang HY, Ding TL, Xie Y, Xu XP, Yu L, Chen T. Osteogenic and adipogenic differentiation of bone marrow-derived mesenchymal stem cells in patients with aplastic anemia. Zhonghua Nei Ke Za Zhi. 2009;48:39–43.

[158]. [158]Wu X, Li Y, Zhu K, Wang Z, Chen S, Yang L. GATA-1, -2 and -3 genes expression in bone marrow microenviroment with chronic aplastic anemia. Hematology. 2007;12:331–335.

[159]. [159]Bacigalupo A, Valle M, Podesta M, Pitto A, Zocchi E, De Flora A, et al. T-cell suppression mediated by mesenchymal stem cells is deficient in patients with severe aplastic anemia. Exp Hematol. 2005;33:819–827. CrossRef

[160]. [160]Chen J, Lipovsky K, Ellison FM, Calado RT, Young NS. Bystander destruction of hematopoietic progenitor and stem cells in a mouse model of infusion-induced bone marrow failure. Blood. 2004;104:1671–1678. MEDLINE | CrossRef

[161]. [161]Scopes J, Ismail M, Marks KJ, Rutherford TR, Draycott GS, Pocock C, et al. Correction of stromal cell defect after bone marrow transplantation in aplastic anaemia. Br J Haematol. 2001;115:642–652. MEDLINE | CrossRef

[162]. [162]Fouillard L, Bensidhoum M, Bories D, Bonte H, Lopez M, Moseley AM, et al. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia. 2003;17:474–476. MEDLINE | CrossRef

[163]. [163]Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res. 2004;95:343–353. CrossRef

[164]. [164]Schatteman GC, Awad O. Hemangioblasts, angioblasts, and adult endothelial cell progenitors. Anat Rec A Discov Mol Cell Evol Biol. 2004;276:13–21. MEDLINE

[165]. [165]Fureder W, Krauth MT, Sperr WR, Sonneck K, Simonitsch-Klupp I, Mullauer L, et al. Evaluation of angiogenesis and vascular endothelial growth factor expression in the bone marrow of patients with aplastic anemia. Am J Pathol. 2006;168:123–130. MEDLINE | CrossRef

[166]. [166]Gupta P, Khurana N, Singh T, Gupta D, Dhingra KK. Bone marrow angiogenesis in aplastic anemia--a study of CD 34 and VEGF expression in bone marrow biopsies. Hematology. 2009;14:16–21.

[167]. [167]Mesa RA, Hanson CA, Rajkumar SV, Schroeder G, Tefferi A. Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia. Blood. 2000;96:3374–3380. MEDLINE

[168]. [168]Aguayo A, Kantarjian H, Manshouri T, Gidel C, Estey E, Thomas D, et al. Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes. Blood. 2000;96:2240–2245. MEDLINE

[169]. [169]Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science. 2007;318:1920–1923. CrossRef

[170]. [170]Ye L, Chang JC, Lin C, Sun X, Yu J, Kan YW. Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases. Proc Natl Acad Sci U S A. 2009;106:9826–9830. CrossRef

[171]. [171]Raya A, Rodriguez-Piza I, Guenechea G, Vassena R, Navarro S, Barrero MJ, et al. Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Nature. 2009;460:53–59. CrossRef

Dr. Jian Ping Li achieved his medical doctoral degree in 1999 and his master degree in 2005 from Qinghai Medical College, Qinghai, China. From 2005 to 2007, he was a member of Department of Hematology at the Affiliated Hospital of Qinghai University. He is currently working as a M.D. fellow in the Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College. His main research focuses on the biology and clinical potential of stem/progenitor cells.

Dr. Cui Ling Zheng achieved her medical doctoral degree in 2002 from Shangdong University, China. From 2002 to 2004, she worked in the Blood Center of Jiangxi Province. She is currently working as a Ph.D. fellow in the Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College. Her research focuses on the differentiation of hematopoietic stem cells and mesenchymal stem cells. She has published several papers in English and in Chinese.

Dr. Zhong Chao Han graduated from Jiangxi Medical College in 1982, from Fujian Medical College as a Master in Medical Sciences in 1984, and achieved his Ph.D. degree in Life Science from Occidental Bretagne University School of Medicine of Brest, France. From 1990 to 1997, he was research scientist and laboratory chief of the Institut des Vaisseaux et du Sang, Paris and then professor associated with university (Hematology) in Paris 7th University. From 1997 to 2004, he was the Director of the Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, and also was the director of the State Key Laboratory for Experimental Hematology. Dr. Han is currently the Director of the National Research Center for Stem Cell Engineering and Technology. Dr. Han has published more than 100 papers in peer-reviewed journals and several books.

State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, PR China

Corresponding author at: State Key Laboratory of Experimental Hematology, National Research Center for Stem Cell Engineering and Technology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, PR China. Tel.: +86 22 27210717; fax: +86 22 66211430.

PII: S1040-8428(09)00246-7

doi:10.1016/j.critrevonc.2009.12.001

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