Effect of thrombopenia on bone marrow CFU-S level and its capability to modify the circulating thrombocyte number

Thrombocytopenia (rise of the thrombopoietin level) was induced by an antithrombocyte serum in mice. After 6 hours of existence of thrombocytopenia, the CFU-S and megakaryocyte-commmitted stem cell content of the bone marrow and spleen was determined by transplantation into mice pretreated with 800 cGy-rtg irradiation. Thrombocytopenia did not influence the CFU-S content. Thrombocytopoiesis of the recipient mice was better restored by bone marrow and spleen cells of thrombocytopenic animals than by cells transplanted from animals with a normal thrombocyte count.     

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.     

Suppressive Effects of Anthrax Lethal Toxin on Megakaryopoiesis

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34⁺ cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax.     

Essential role for cyclin D3 in granulocyte colony-stimulating factor-driven expansion of neutrophil granulocytes

The proliferation of neutrophil granulocyte lineage is driven largely by granulocyte colony-stimulating factor (G-CSF) acting via the G-CSF receptors. In this study, we show that mice lacking cyclin D3, a component of the core cell cycle machinery, are refractory to stimulation by the G-CSF. Consequently, cyclin D3-null mice display deficient maturation of granulocytes in the bone marrow and have reduced levels of neutrophil granulocytes in their peripheral blood. The mutant mice are unable to mount a normal response to bacterial challenge and succumb to microbial infections. In contrast, the expansion of hematopoietic stem cells and lineage-committed myeloid progenitors proceeds relatively normally in mice lacking cyclin D3, revealing that the requirement for cyclin D3 function operates at later stages of neutrophil development. Importantly, we verified that this requirement is specific to cyclin D3, as mice lacking other G(1) cyclins (D1, D2, E1, or E2) display normal granulocyte counts. Our analyses revealed that in the bone marrow cells of wild-type mice, activation of the G-CSF receptor leads to upregulation of cyclin D3. Collectively, these results demonstrate that cyclin D3 is an essential cell cycle recipient of G-CSF signaling, and they provide a molecular link of how G-CSF-dependent signaling triggers cell proliferation.     

Granulocyte-dendritic cell unbalance in the non-obese diabetic mice

Several investigators, including ourselves, have reported lower yield of GM-CSF bone marrow-derived dendritic cells (DC) with altered MHC class II and co-stimulatory molecules expression in the non-obese diabetic (NOD) mice. However, whether this defect was intrinsic to the DC lineage and/or related to abnormal expansion of other cell types responding to GM-CSF remained an opened issue. We performed phenotypical and morphological analysis of cells from GM-CSF-supplemented-bone marrow-cultures and of freshly isolated bone marrow and blood cells from unmanipulated prediabetic NOD mice. The results show a heretofore undescribed bias towards generation of granulocytes in NOD mice, concomitant with quantitative and qualitative alterations of the DC lineage in both the bone marrow and the blood of this mouse strain. We propose that increased generation of granulocytes in NOD mice might contribute to autoimmunity. First, high numbers of granulocytes per se might favor inflammatory environment. Second, granulocytes, by interfering with DC development, might favor unbalanced antigen presenting cell function leading to T cell autoimmunity.     

Changes in hemopoietic and regulator levels in mice during fatal or nonfatal malarial infections. II. Nonerythroid populations

Levels of mature lymphocytes, granulocytes, macrophages, platelets, their progenitor cells, and cytokines were monitored in the blood, marrow, and spleen during fatal or nonfatal murine malarial infections. In all four malaria models, before anemia developed, there was a lymphopenia, a rapid lymphocyte depletion in the marrow with a compensating rise in spleen lymphocytes, thrombocytopenia with increased megakaryocytic progenitor cell numbers, and monocyte increases in the bone marrow and later the spleen. The development of anemia was associated with a monocytosis and neutropenia, an increase in granulomonocytic progenitor cells in the spleen, and a reduction of spleen lymphocytes. Spleen granulocytes, monocytes, and their progenitor cells increased two- to threefold more in nonfatal than in fatal malaria and the spleen lymphocyte pool became severely depleted in fatal malaria. The data suggest that a defective effector cell response was of importance for the fatal outcome of the disease. Other than an early rise in serum macrophage colony stimulating factor levels in fatal infections, changes in levels of the regulators of these effector cells did not correlate well with the outcome of the infection.     

Panax ginseng modulates cytokines in bone marrow toxicity and myelopoiesis: ginsenoside Rg1 partially supports myelopoiesis

In this study, we have demonstrated that Korean Panax ginseng (KG) significantly enhances myelopoiesis in vitro and reconstitutes bone marrow after 5-flurouracil-induced (5FU) myelosuppression in mice. KG promoted total white blood cell, lymphocyte, neutrophil and platelet counts and improved body weight, spleen weight, and thymus weight. The number of CFU-GM in bone marrow cells of mice and serum levels of IL-3 and GM-CSF were significantly improved after KG treatment. KG induced significant c-Kit, SCF and IL-1 mRNA expression in spleen. Moreover, treatment with KG led to marked improvements in 5FU-induced histopathological changes in bone marrow and spleen, and partial suppression of thymus damage. The levels of IL-3 and GM-CSF in cultured bone marrow cells after 24 h stimulation with KG were considerably increased. The mechanism underlying promotion of myelopoiesis by KG was assessed by monitoring gene expression at two time-points of 4 and 8 h. Treatment with Rg1 (0.5, 1 and 1.5 μmol) specifically enhanced c-Kit, IL-6 and TNF-α mRNA expression in cultured bone marrow cells. Our results collectively suggest that the anti-myelotoxicity activity and promotion of myelopoiesis by KG are mediated through cytokines. Moreover, the ginsenoside, Rg1, supports the role of KG in myelopoiesis to some extent.     

Inhibition of Megakaryocyte Development in the Bone Marrow Underlies Dengue Virus-Induced Thrombocytopenia in Humanized Mice

A characteristic clinical feature of dengue virus infection is thrombocytopenia, though its underlying mechanism is not definitively determined. By adoptive transfer of human CD34⁺ fetal liver cells into immunodeficient mice, we have constructed humanized mice with significant levels of human platelets, monocytes/macrophages, and hepatocytes. Infection of these mice with both lab-adapted and clinical strains of dengue virus induces characteristic human hematological changes, including transient leukopenia and thrombocytopenia. We show that the specific depletion of human platelets is not mediated by antibodies in the periphery or reduced production of human thrombopoietin in the liver but reduction of human megakaryocytes and megakaryocyte progenitors in the bone marrow of the infected mice. These findings identify inhibition of platelet production in the bone marrow as a key mechanism underlying dengue-induced thrombocytopenia and suggest the utility of the improved humanized mouse model in studying dengue virus infection and pathogenesis in a human cell context.     

Constitutive expression of IL-6-LIKE cytokines in normal bone marrow: implications for pathophysiology of myeloma

Myeloma is a neoplasm thought to "home" to bone marrow. However, evidence for bone-marrow-specific receptors or adhesion molecules expressed on myeloma cells is scanty. Initial myeloma expansion is thought to be due to IL-6 and/or related cytokines. Previous determinations of cytokine expression in bone marrow were performed on bone marrow stromal lines; these findings may not reflect the constitutive pattern of expression in situ. Intracytoplasmic staining for IL-6-like cytokines revealed constitutive expression of some factors in the bone marrow of normal mice, but not spleens. Spleens of myeloma-transplanted SCID mice expressed IL-6-like cytokines, indicative of induction of expression by myeloma. Some cytokines expressed in bone marrow induced myeloma proliferation in the presence of dexamethasone, demonstrating dependence of the myeloma on these cytokines. Our data imply that, rather than "homing" to bone marrow, myeloma cells proliferated within marrow cavities more than in other organs because of growth factors constitutively expressed by bone marrow cells. As myeloma progressed, we observed the induction of growth factor expression in spleen cells. Furthermore, because cytokines other than IL-6 may induce myeloma cell proliferation, therapy aimed at neutralizing IL-6 may not be the most effective method to treat this disease. These findings have implications for both the pathophysiology and therapy of multiple myeloma.     

Effects of hypoxia on the small acetylcholinesterase-positive megakaryocyte precursor in bone marrow of mice

The number of small acetylcholinesterase-positive (SAChE+) cells in the marrow of hypoxic mice was measured. Mice were exposed to 6-7% O2 levels by enclosure in cages covered with dimethyl-silicone rubber membranes for 1-14 days. The mice showed a linear increase in packed cell volumes with time in the hypoxic atmosphere, but platelet counts showed a characteristic biphasic response, i.e., increased platelet counts were observed after 1-3 days of hypoxia, and significantly (P less than 0.05-P less than 0.0005) decreased platelet counts were observed thereafter (6-14 days). The total number of megakaryocytes in the marrow of hypoxic mice decreased significantly (P less than 0.005) with time. In agreement with the data on platelet counts, hypoxia caused the total number of SAChE+ cells in the marrow of mice to be biphasic. At Day 2 there was a significant increase (P less than 0.05) in the total number of SAChE+ cells/mm3 of bone marrow; however, by days 10-14 the numbers had decreased markedly (P less than 0.005). These data indicate that hypoxia decreases platelet production by action on a precursor cell to the SAChE+ cell. The hypoxia-induced thrombocytopenia is probably caused by stem-cell competition between the erythrocytic and megakaryocytic cell lines.     

Effect of pregnancy on serum alanine concentration in normal and genetically diabetic mice.

Serum alanine concentration was determined in nonpregnant and pregnant normal control Swiss albino (SA) and genetically diabetic KK mice. The serum alanine levels were significantly lower in nonpregnant KK than in nonpregnant SA mice. Fasting elicited hypoglycemia, hypoinsulinemia and hypoalaninemia in both groups of pregnant mice. Oral administration of alanine in nonpregnant and pregnant mice resulted in a significant rise in blood sugar levels within 15 min in both groups. However, the initial blood sugar response to oral alanine was greater in pregnant than in nonpregnant mice. This increase in blood sugar response to exogenous alanine appears to be mediated by glucagon. The data suggest that pregnancy elicits hypoglycemia, hypoinsulinemia and hypoalaninemia in both nondiabetic and diabetic mice.     

Smad3基因剔除对小鼠造血功能的影响

研究Smad3基因剔除对小鼠造血功能的影响。实验小鼠分为 5组 ,每组有Smad3基因剔除小鼠(Smad3 - - )和其同窝孪生的野生型小鼠 (Smad3 + + )各 1只。小鼠的造血功能用 14天形成的脾结节 (CFU S1 4 )、多系祖细胞 (CFU GEMM)、粒 单系祖细胞 (CFU GM)、红系祖细胞 (BFU E)测定及外周血象、骨髓象等实验血液学指标来确定。每组小鼠取尾血作白细胞、红细胞和血小板计数 ,涂片作白细胞分类计数。将一侧股骨的骨髓冲出 ,制成单细胞悬液 ,计数其中有核细胞数 ,测定CFU GM、BFU E、CFU GEMM值。将每只小鼠的 4× 10 4个骨髓有核细胞 ,经尾静脉注入 3只 8～ 10周经致死量射线照射的同系雌性小鼠体内 ,测定 14天的CFU S。取一部分胸骨、肝脏、脾脏固定做病理切片 ,其余胸骨冲出骨髓 ,涂片作分类计数。结果Smad3 - - 小鼠外周血白细胞和血小板计数明显高于Smad3 + + 小鼠 ,红细胞数无显著差异。外周血白细胞分类结果也表明粒细胞显著增高。骨髓有核细胞数无显著差异 ,CFU GM显著增高 ,BFU E无显著差异 ,CFU GEMM明显减少 ,CFU S显著减少。病理形态学观察发现骨髓增生极度活跃 ,以粒系为主 ,肝脾无显著差别。骨髓涂片分类表明粒系增多 ,粒系 :红系比例增高。因此得出结论Smad3基因剔除使小鼠造血干祖细胞数目     

Multiple levels of regulation of megakaryocytopoiesis

A working hypothesis for the regulation of megakaryocytopoiesis is described on the basis of current data. The hypothesis proposes that in vivo megakaryocytes are generated by 1) the expansion of clonable progenitor cells into immature megakaryocytes by locally produced (and regulated) interleukin-3 (IL-3) and 2) the development and maturation of immature megakaryocytes by a dual system; by a lineage specific mechanism involving thrombopoietic stimuli in the steady state and thrombocytopenic conditions, and by a lineage nonspecific mechanism via IL-3 in damaged or reconstituting marrow. The hypothesis predicts that if IL-3 is a significant in vivo regulator of megakaryocyte formation and development, receptor for IL-3 should be present on megakaryocytes and may be vestigially on platelets. Small but significant levels of 125I IL-3 were found to bind to platelets from normal mice. The level of binding on platelets was found to be enhanced sevenfold from mice that had received high levels of irradiation followed by bone marrow transplantation. This contrasted with a twofold increase in the level of binding to platelets from mice made acutely thrombocytopenic with antiplatelet serum. The data suggest that IL-3 may be involved in the in vivo regulation of murine megakaryocytopoiesis and may be a significant factor in rebound thrombopoiesis following bone marrow damage.     

Platelet Changes in Acute Leukemia

Acute leukemia is a hematopoietic stem cell malignant disease, with abnormal proliferation of leukemic and immature cells that suppress the production of normal blood cells and extensively invade peripheral tissues. The bleeding complications are very common in acute leukemia and often lead to death. One major cause for hemorrhage is thrombocytopenia, which is caused by the replacement of normal bone marrow cells with leukemic cells and the inhibition of megakaryocytes functions. Declines in platelet count as well as in function in acute leukemia have been reported in many studies. Here, we reviewed the literatures concerning platelet changes in acute leukemia.     

Immune thrombocytopenic purpura associated with hepatitis A

A 23-year-old man developed thrombocytopenic purpura at the end of the second week of the clinical evolution of hepatitis A confirmed by viral markers. The bone marrow of this patient showed megakaryocytic hyperplasia. Circulating in his serum immune complexes were demonstrated by solid phase conglutinin enzymo-immunoassay. Platelet-reactive serum factors were also detected by an indirect immunofluorescence test using fresh donor platelets as targets. The evolution of both the hepatitis and the purpura were benign with no therapy other than bedrest. Platelet count normalized within five weeks of the onset of purpura, and IgM antibodies against hepatitis A virus as well as circulating immune complexes dropped to normal levels. It is postulated that the thrombocytopenia of this case was caused by nonspecific deposition of immune complexes at the platelet surface.     

Cordyceps sinensis health supplement enhances recovery from taxol-induced leukopenia

This study aimed to evaluate the ability of the health food supplement Cordyceps sinensis (CS) to ameliorate suppressive effects of chemotherapy on bone marrow function as a model for cancer treatment. Mice were treated with Taxol (17 mg/kg body wt) one day before oral administration of a hot-water extract of CS (50 mg/kg daily) that was given daily for 3 weeks. White blood cell counts in peripheral blood of mice receiving Taxol were at 50% of normal levels on day 28 but had recovered completely in mice treated with CS. In vitro assays showed that CS enhanced the colony-forming ability of both granulocyte macrophage colony forming unit (GM-CFU) and osteogenic cells from bone marrow preparations and promoted the differentiation of bone marrow mesenchymal stromal cells into adipocytes, alkaline phosphatase-positive osteoblasts, and bone tissue. This result could be attributed to enhanced expression of Cbfa1 (core binding factor a) and BMP-2 (bone morphogenetic protein) with concurrent suppression of ODF (osteoclast differentiation factor/RANK [receptor activator of NF-kappaB]) ligand. In summary, CS enhances recovery of mice from leukopenia caused by Taxol treatment. It appears to do so by protecting both hematopoietic progenitor cells directly and the bone marrow stem cell niche through its effects on osteoblast differentiation.     

Distinct human immunodeficiency virus strains in the bone marrow are associated with the development of thrombocytopenia

We analyzed bone marrow and blood from human immunodeficiency virus type 1 (HIV-1)-infected individuals and described the HIV-1 quasispecies in these cellular compartments. HIV-1 isolates from the bone marrow of thrombocytopenic patients contained distinct amino acids in the V3 loop and infected T-cell lines, implicating this virus in the development of thrombocytopenia.     

Association of 5′nucleotidase activity with immature granulocytes in the bone marrow of guinea pigs

Bone marrow leukocytes from adult strain 2 guinea pigs were found to have appreciable levels of 5′adenosine monophosphate hydrolytic activity (105 nmole/h/10⁶ cells). On the basis of substrate specificity studies, enzyme inhibition studies, and thin-layer chromatographic analysis of the reaction product, the activity is related to 5′nucleotidase (5′N). The enzyme activity was associated with the membrane-enriched particulate fraction of lysed bone marrow cells.The bone marrow cell-associated 5′N activity was consistently very high in all five strains of guinea pigs examined (77–127 nmole/h/10⁶ cells) and the range of activity was at least 10-fold greater than that observed for bone marrow cells obtained from mice, rabbits or rats. Furthermore, the bone marrow cell-associated 5′N activity in strain 2 guinea pigs was 5-fold greater than that observed for spleen and at least 13-fold greater than that of blood, mesenteric lymph nodes or thymus obtained from the same animal.Fractionation of strain 2 guinea pig bone marrow cells on Percoll density gradients showed that as the proportion of immature granulocytes increased in the various cell fractions, so did the 5′N activity. The cell fraction that sedimented at a density of 1.071 g/ml had the highest 5′N activity and the majority of the cells (94%) were immature granulocytes. The bone marrow compared to blood and spleen had the highest number of total granulocytes and the highest percentage of immature granulocytes. We conclude that the elevated bone marrow-derived 5′N activity in guinea pigs is associated with the resident population of immature granulocytes in that tissue.     

Effects of cyclophosphamide on murine bone marrow and splenic megakaryocyte-CFC,granulocyte-macrophage-CFC,and peripheral blood cell levels

The effect of cyclophosphamide (CY) on megakaryocytopoiesis in mice was examined with assays of megakaryocyte colony-forming cells (Meg-CFC) in bone marrow and spleen and simultaneous determinations of peripheral blood counts, after a single intraperitoneal dose (200 mg/kg) of CY. Significant rebound thrombocytosis (170% of normal) occurred at day 11 after injection with CY, although only modest preceding thrombocytopenia (70% of normal) was observed. After an initial 3–5-day period of suppression, total megakaryocyte colony-forming cells (Meg-CFC) in both bone marrow and spleen of CY-treated mice demonstrated rebound increases at 5 and 7 days, respectively, after administration of the drug. Granulocyte-macrophage colony-forming cells (GM-CFC) exhibited alterations which were similar to those of Meg-CFC, suggesting similar sensitivities of Meg-CFC and GM-CFC to CY. The increase in Meg-CFC in both bone marrow and spleen preceded development of thrombocytosis by 4–6 days. This suggests that increased platelet counts in CY-treated mice are attributable, at least in part, to alterations in feedback mechanisms which control megakaryocytopoiesis, with resultant stimulation of the megakaryocyte progenitor compartment.