Irradiation enhances the support of haemopoietic cell transmigration, proliferation and differentiation by endothelial cells.

Endothelial cells (ECs) are a critical component of the bone marrow stroma in the regulation of haemopoiesis. Recovery of bone marrow aplasia after radiation exposure depends, in part, on the repair of radiation-induced endothelial damage. Therefore, we assessed the ability of an irradiated human bone marrow EC line (TrHBMEC) to support transmigration, proliferation and differentiation of CD34+ bone marrow cells either irradiated or not in transendothelial migration or co-culture models. Radiation-induced EC damage was reflected by an increased release of soluble intercellular adhesion molecule (sICAM)-1 and platelet endothelial cell adhesion molecule (PECAM)-1. Irradiation of TrHBMECs with a 10 Gy dose strongly enhanced the transmigration of CD34+ cells, granulo-monocytic progenitors (CFU-GM) and erythroid progenitors (BFU-E). While ICAM-1 and PECAM-1 expression on irradiated TrHBMECs was increased, only antibodies against PECAM-1 inhibited the radiation-induced enhanced transmigration of haemopoietic cells. Irradiation of TrHBMECs (5-15 Gy) also increased proliferation and differentiation towards the granulo-monocytic lineage of co-cultured CD34+ cells, as well as colony formation by those cells and the production of interleukin 6 (IL-6), IL-8, granulocyte colony-stimulating factor (CSF) and granulocyte-macrophage CSF. Irradiated TrHBMECs were more capable of stimulating irradiated (1,2 Gy) CD34+ cells and haemopoietic progenitors than non-irradiated TrHBMECs. Together, these results suggest that, despite the radiation-induced damage, irradiated ECs may favour haemopoietic reconstitution after radiation exposure.

Inflammatory response to abdominal irradiation stimulates hemopoiesis.

PURPOSE: The connection between inflammation and hemopoiesis was studied in the context of abdominal irradiation. MATERIALS AND METHODS: Male C57BL6/J mice received localized irradiation of 20 Gy either to 70% of the liver or to the intestine (most of ileum and caecum). RESULTS: Irradiation of liver induced a rapid increase in intercellular adhesion molecule-1 mRNA expression in the liver. In serum/plasma, an increase in positive acute phase proteins (serum amyloid-P and fibrinogen) and a decrease in albumin occurred during the second and third week following liver irradiation. Similarly, intestinal irradiation induced an increase in plasma fibrinogen level. A transient elevation in neutrophil and platelet counts was observed that was maximal during the second and third week with similar kinetics for intestinal and liver irradiation. Moreover, intestinal irradiation enhanced hemopoietic progenitors in bone marrow. IL-6, which is known to be an agonist in the regulation of acute phase protein expression as well as hemopoietic cell production, was increased in plasma from intestinal- and liver-irradiated mice. Administration of an anti-IL-6 mAb to intestinal-irradiated mice abrogated the elevation of fibrinogen and the increase in hemopoietic progenitors. CONCLUSIONS: Abdominal irradiation provokes an inflammatory response which in turn stimulates hemopoiesis. IL-6 may play a major role in controlling these events.

Single administration of thrombopoietin to lethally irradiated mice prevents infectious and thrombotic events leading to mortality.

A sufficiently high dose of thrombopoietin to overcome initial c-mpl-mediated clearance stimulates hematopoietic reconstitution following myelosuppressive treatment. We studied the efficacy of thrombopoietin on survival after supralethal total body irradiation (9 Gy) of C57BL6/J mice and the occurrence of infectious and thrombotic complications in comparison with a bone marrow graft or prophylactic antibiotic treatment. Administration of 0.3 microg thrombopoietin, 2 hours after irradiation, protected 62% of the mice as opposed to no survival in placebo controls. A graft with a supraoptimal number of syngeneic bone marrow cells (10(6) cells) fully prevented mortality, whereas antibiotic treatment was ineffective. Blood cell recovery was observed in the thrombopoietin-treated mice but not in the placebo or antibiotic-treated group. Bone marrow and spleen cellularity as well as colony-forming unit granulocyte-macrophage and burst-forming unit erythroid were considerably increased in thrombopoietin-treated mice relative to controls. Histologic examination at day 11 revealed numerous petechiae and vascular obstructions within the brain microvasculature of placebo-treated mice, which was correlated with hypercoagulation and hypofibrinolysis. Thrombopoietin treatment prevented coagulation/fibrinolysis disorder and vascular thrombosis. High fibrinogen levels were related to bacterial infections in 67% of placebo-treated mice and predicted mortality, whereas the majority of the thrombopoietin-treated mice did not show high fibrinogen levels and endotoxin was not detectable in plasma. We conclude that thrombopoietin administration prevents mortality in mice subjected to 9-Gy total body irradiation both by interfering in the cascade leading to thrombotic complications and by amelioration of neutrophil and platelet recovery and thus protects against infections and hemorrhages.

Interleukin 4 promotes survival of lethally irradiated mice in the absence of hematopoietic efficacy.

The therapeutic potential of Il4 in lethally irradiated mice was evaluated in C57BL6/J mice subjected to 7 to 10 Gy total-body irradiation (TBI) from a (60)Co gamma-ray source. Il4 was administered 2 h after TBI either in a single injection or for 5 consecutive days. Il4 treatment increased 30-day survival of mice irradiated with doses as high as 8.5 Gy, which caused 100% mortality in placebo-treated animals. By convention, hematopoietic failure would induce death over a period of up to 30 days. However, in our study, the Il4-enhanced survival of mice within this period could not be attributed to significantly accelerated hematopoietic reconstitution as shown by blood cell counts and progenitor cell contents in the bone marrow and spleen. Our data strongly suggest that aplasia is not the only cause of death of animals irradiated with doses around the LD(50) and that Il4-treated animals can survive in spite of a very poor hematopoietic activity.

Preferential liver irradiation enhances hematopoiesis through a thrombopoietin-independent mechanism.

Liver synthesizes thrombopoietin, which is a major cytokine involved in the production of hematopoietic cells. The purpose of this study was to examine the effects of preferential liver irradiation on expression of thrombopoietin and production of hematopoietic cells. About 70% of the liver of C57BL6/J mice was irradiated with 20 Gy of gamma rays. Exposure to ionizing radiation enhanced hematopoietic progenitors and megakaryocyte frequency in bone marrow and induced a transient increase in platelet and neutrophil counts that peaked 14 days after irradiation. The concentration of thrombopoietin was increased in serum as early as 5 h after liver irradiation and was still elevated at day 14. By using Northern blot analysis and an RNase protection assay, we showed that thrombopoietin mRNA was increased in the irradiated liver. To determine whether thrombopoietin was involved in the stimulation of hematopoiesis, we irradiated mice in which thrombopoietin deficiency had been induced by homologous recombination. Platelet levels were increased in both heterozygous and homozygous thrombopoietin-deficient mice with a magnitude similar to that obtained in normal mice. In summary, our data demonstrate that local irradiation of the abdomen encompassing the liver leads to stimulation of hematopoiesis through a thrombopoietin-independent mechanism.

Thrombopoietin promotes hematopoietic recovery and survival after high-dose whole body irradiation.

PURPOSE: The therapeutic potential of thrombopoietin (TPO), the major regulator of platelet production, was evaluated for hematopoietic recovery and survival in mice following lethal and supralethal total body irradiation (TBI). METHODS AND MATERIALS: Hematopoietic recovery was studied in C57BL6/J mice after 8 Gy TBI (gamma-rays). Survival experiments were performed with C57BL6/J and BCBA F1 mice. Two protocols of TPO administration were evaluated: treatment for 7 consecutive days (7 x 0.3 microg/mice) beginning 2 h after exposure, or a single dose (0.3 microg/mice) administered 2 h after irradiation. RESULTS: TPO improved the platelet nadir and accelerated the platelet reconstitution of irradiated mice in comparison to placebo-treated mice. Recovery of neutrophils and erythrocytes was stimulated as well. TPO induced an accelerated recovery of hematopoietic progenitors and immature multilineage progenitors in bone marrow and spleen. In addition, TPO administration induced approximately 90% survival of 8 Gy irradiated C57BL6/J mice, a TBI dose which resulted in 100% mortality within 30 days for placebo-treated mice. Single TPO administration was as effective as repeated injections for hematopoietic recovery and prevention of mortality. Dose-effect survival experiments were performed in BCBA F1 mice and demonstrated that TPO shifted the LD50/30 from approximately 9.5 Gy to 10.5 Gy TBI given as a single dose, and from 14 Gy to as high as 17 Gy when TBI was given in three equal doses, each separated by 24 h. CONCLUSION: These results demonstrate that the multilineage hematopoietic effects of TPO may be advantageously used to protect against lethal bone marrow failure following high dose TBI.

Late and persistent up-regulation of intercellular adhesion molecule-1 (ICAM-1) expression by ionizing radiation in human endothelial cells in vitro.

Adhesion molecules play a key role in cellular traffic through vascular endothelium, in particular during the inflammatory response when leukocytes migrate from blood into tissues. Since inflammation is one of the major consequences of radiation injury, we investigated the effect of ionizing radiation on cell-surface expression of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in cultured human umbilical vein endothelial cells (HUVEC). Flow cytometry performed on irradiated HUVEC revealed both a time- (from 2 to 10 days) and dose- (from 2 to 10 Gy) dependent up-regulation of basal expression of ICAM-1, and no induction of VCAM-1 or E-selectin. The radiation-induced increase in ICAM-1 expression on HUVEC was correlated with augmented adhesion of neutrophils on irradiated endothelial cells. Interleukin-6 (Il-6) or other soluble factors released by irradiation were not involved in the enhanced ICAM-1 expression by irradiation. Northern blot analysis showed an overexpression of ICAM-1 mRNA from 1 to 6 days after a 10 Gy exposure. Our data suggest that ICAM-1 participates in the radiation-induced inflammatory reaction of the endothelium.

Growth and differentiation of the human megakaryoblastic cell line (ELF-153): a model for early stages of megakaryocytopoiesis.

ELF-153 is a cell line that has been established from a patient with a poorly differentiated acute myeloid leukemia associated with an acute myelofibrosis. A majority of cells had a blast morphology with the phenotype of a myeloid hematopoietic progenitor, ie, CD34+, CD33+, CD13+, HLA-DR+, but CD38-, and the remaining cells (5% to 10%) expressed platelet restricted proteins such as CD41, CD42, CD36, CD61, and von Willebrand factor; some of them were polyploid (up to 32N) and exhibited demarcation membranes and alpha granules. No erythroid or other lineage-specific markers were detected. Proliferation of ELF-153 cells was highly stimulated by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor and to a lesser extent by stem cell factor and IL-6. In contrast, the cell line did not respond to erythropoietin, leukemia inhibitory factor, IL-7, IL-11, granulocyte colony-stimulating factor, and basic fibroblast growth factor. ELF-153 cells could be separated by flow cytometry into three discrete cell populations (CD34+/CD61-, CD34+/CD61+, and CD34-/CD61+) with different proliferative and endomitotic properties corresponding to distinct stages of the mega karyocyte (MK) differentiation. This MK differentiation, which involved a minority of ELF-153, could be increased in the presence of 5-azacytidine and phorbol ester, but could not be significantly modified by growth factors. By contrast, cytochalasin B dramatically induced polyploidization without differentiation. It is noteworthy that association of 5-azacytidine to cytochalasin B dramatically induced the production of polyploid MK cells. To understand the molecular mechanisms underlying this MK differentiation, the expression of GATA-1 and GATA-2 was investigated in subpopulations of ELF-153. A high level of GATA-1 and GATA-2 mRNA was only present in the CD61+ cells. Therefore, these two transactivating factors may play an important role in the MK differentiation of ELF-153. We conclude that ELF-153 might be an important tool to investigate the mechanisms by which transcription factors control differentiation of MK progenitors.

[Hematopoiesis and its regulation. Comparison between erythropoiesis and megakaryocytopoiesis]. / Hématopoïèse et sa régulation. Comparaison entre l'érythropoïèse et la mégacaryocytopoïèse.

Hematopoiesis is the cellular system which leads to the continuous production of blood cells. This highly complex cellular system is organized into three main compartments: (i) stem cells which are both pluripotent and theoretically capable of self renewal; (ii) hematopoietic progenitors which are committed to (only) one cell lineage and are able to proliferate along each particular differentiation pathway; (iii) a maturation compartment in which cells become morphologically identifiable since they synthesize lineage specific proteins. The maturation cell compartment represents the majority of marrow cells. At the present time, the regulation of true stem cells remains poorly understood since these cells are difficult to assay in vitro. In contrast, the regulation of each hematopoietic lineages becomes to be well known. These knowledges are mainly due to two reasons: (i) hematopoietic progenitors can be purified and assayed in culture. Their proliferation and differentiation are strictly dependent upon the presence of hematopoietic growth factors; (ii) these different hematopoietic growth factors have been isolated and their cDNA cloned. Erythropoiesis and megakaryocytopoiesis are two branches of hematopoiesis which lead to the production of RBC and platelets, respectively. These two cell lineages have several common features. However, they markedly differ by their regulation since RBC production depends upon one main stimulus (hypoxia) and, therefore, the terminal erythroid differentiation is regulated by a single growth factor. In contrast, regulation of platelet production may depend on several stimuli such as the platelet mass (homeostasis), inflammation, infection and hypoxia. Therefore, several cytokines are involved in the regulation of megakaryocytopoiesis. In addition, the mechanisms of platelet production are highly complex and, in contrast to all the other hematopoietic lineages where the production of mature cells depends on a single parameter (the proliferation during differentiation), three independent parameters modify thrombopoiesis: a) the number of marrow megakaryocytes (MK) (proliferation of the precursor cells). b) the megakaryocyte volume which directly depends on the MK ploidy. During MK differentiation, MK precursors switch from a mitotic process (DNA duplication followed by cytokinesis) to an endomitotic process (DNA duplication without cytokinesis). Endomitosis is a specific process of the megakaryocytic differentiation and differs from all the other cellular models of polyploidization by the existence of a single polyploid and polylobulated nucleus in each cell. This polyploidization induces a major amplification of the platelet production since it is associated with a parallel increase in the cytoplasmic mass.(ABSTRACT TRUNCATED AT 400 WORDS)

Erroneous results of 3H-thymidine incorporation are related to position of thymidine residues in oligodeoxynucleotides.

Antisense oligodeoxynucleotides (ODNs) targeted to complementary mRNA sequences have proved to be a powerful approach in assessing the function and the role of unique genes in cell proliferation, differentiation, or transformation. Despite their importance in the development of future therapies, little is known about their fate after uptake by cells. Here, we have examined the contribution of individual nucleotide residues from synthetic nonspecific ODNs on assays commonly used to measure cell proliferation. A dramatic decrease of the 3H-thymidine (3H-T) incorporation was obtained with nonspecific ODNs, while no effect on cell proliferation was observed as assessed by three other techniques. We demonstrate that the presence and position of thymidine in the ODNs directly interfere with the intracellular thymidine pool, leading to faulty data of 3H-T incorporation. As an alternative method, we used 3H-deoxyuridine (3H-dU), which is integrated more efficiently in DNA than thymidine. We observed that 3H-dU incorporation was also decreased. In conclusion, caution should be exercised in the interpretation of the results of 3H-T and 3H-dU incorporation in the presence of oligodeoxynucleotides.

Expression of tal-1 and GATA-binding proteins during human hematopoiesis.

Tal-1 rearrangements are associated with nearly 30% of human T acute lymphoblastic leukemia. Tal-1 gene encodes a putative transcription factor with a basic helix-loop-helix domain and is known to be predominantly expressed in hematopoietic cells. We investigated the pattern of tal-1 expression in purified human hematopoietic cells by in situ hybridization and reverse transcriptase polymerase chain reaction analysis. Both methods demonstrated that the tal-1 gene is expressed in megakaryocytes and erythroblasts as well as in basophilic granulocytes. In addition, our results indicate that the tal-1 1A promoter, which contains two consensus GATA-binding sites, is active mainly in these lineages. Because the GATA-1 gene is known to transactivate several genes specific for the erythroid, megakaryocytic, and mastocytic/basophilic lineages, we studied GATA-1 expression in these purified hematopoietic cells. We found that GATA-1 and tal-1 genes are coexpressed in these three lineages. Remarkably, the expression of both genes is downmodulated during erythroid and megakaryocytic terminal maturation. In immature hematopoietic cells, tal-1 and GATA-1 genes are coexpressed in committed progenitors cells (CD34+/CD38(2+)), whereas they are not detectable in the most primitive cells (CD34(2+)/CD38-). In contrast, GATA-2 is strongly expressed in both most primitive and committed progenitors cells, whereas GATA-3 is mostly detected in most primitive ones. Altogether our results strongly suggest that GATA-1 modulates the transcription of tal-1 during the differentiation of the erythroid, megakaryocytic, and basosophilic lineages.

c-jun and c-fos are expressed by human megakaryocytes.

Expression of the main nuclear protooncogenes during terminal megakaryocyte (MK) differentiation is poorly understood. Because previous results have suggested that c-fos and c-jun protooncogenes are expressed in human leukemic cell lines induced to undergo megakaryocytic differentiation, we have analyzed the expression of these two protooncogenes in normal MK. Studies were performed, by in situ hybridization and immunofluorescence, on human MK obtained either directly from bone marrow or from culture of MK progenitors. c-fos and c-jun transcripts were detected in most cultured or fresh marrow MK from adult donors. Expression was much higher in cytologically immature than in mature MK whereas no expression was detected in the most mature MK. c-fos and c-jun expression increased dramatically with MK size. In cultured fetal MK, which all remained small in size, c-fos mRNA was present but at a low level. The c-fos-encoded protein (P62fos) was easily detectable in the great majority of MK. We directly demonstrated that the level of P62fos expression was correlated to MK ploidy by flow cytometry using a three-color staining technique. The involvement of serum and growth factors in the induction of P62fos in MK was studied. Whereas a 3-h serum deprivation resulted in the disappearance of P62fos in MK, several growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 7 (IL-7), leukemia inhibitory factor (LIF), and transforming growth factor beta (TGF-beta), as well as normal or aplastic serum, were able to reinduce its expression within 2 h. In conclusion, our results suggest that c-jun and c-fos may play a role in the transduction of signals by several growth factors during terminal MK differentiation.

In vitro effects of hematopoietic growth factors on the proliferation, endoreplication, and maturation of human megakaryocytes.

A liquid culture technique was used to study regulation of human megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow were cultured in the presence of normal plasma, plasma from patients with aplastic marrows (AP), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3). Megakaryocytes (MK) were studied at day 10 of culture by a two-color staining technique using a pool of monoclonal antibodies for their identification and propidium iodide to label DNA. Their ploidy distribution was analyzed by flow cytometry. In some experiments cytoplasmic maturation was also studied by ultrastructural techniques. Normal plasma provides a low number of MK with a ploidy distribution including 8 N and 16 N MK. AP promoted in a dose-dependent manner proliferation of MK and some batches favored endoreplication. This effect was clearly demonstrated when ploidy distribution was compared between normal plasma and AP on parallel marrow cultures. However, ploidy distribution was shifted toward low values compared with uncultured MK. rhGM-CSF had no significant effect on these two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a proliferative effect but was unable to induce endoreplication. Furthermore, when associated with AP it totally abrogated the effect of AP on endoreplication because in most experiments more than 90% of MK were 2 N and 4 N. This effect was also observed when rhIL-3 was added after 7 days of culture (when it has little proliferative effects). Studies of the maturation of MK grown with rhIL-3 indicate that the majority were small mature cells synthesizing alpha-granules and demarcation membranes. The effect of AP on MK proliferation and endoreplication was not related to IL-6 because its IL-6 content was identical to that of normal plasma and its neutralization did not modify these parameters. In conclusion, this study indicates that liquid culture technique in association with flow cytometry could be a powerful tool in identifying the humoral regulators of human megakaryocytopoiesis.

Regulation of human megakaryocytopoiesis: analysis of proliferation, ploidy and maturation in liquid cultures.

A liquid culture technique associated with either double staining and flow cytometry or electron microscopy was used to study human megakaryocytopoiesis. During development from the embryo to the adult, a progressive increase in ploidy classes associated with an enhancement of megakaryocyte (meg) size was observed. Granulocyte-macrophage colony-stimulating factor had no effects on adult marrow cultures. In contrast, interleukin (IL) 3 induced a marked proliferation, but was unable to promote polyploidization. Furthermore, it abrogated the effects on endomitosis of aplastic plasma (AP). This negative effect on polyploidization of IL-3 could be partially dissociated from its effects on proliferation by a delayed addition in culture. AP acted on both proliferation and endoreplication, which was not due to the main hematopoietic growth factors, including IL-6. A synthesis of IL-6 was detected by in situ hybridization in cultured cells including megs which also express receptors for IL-6. These results suggest that terminal meg differentiation may be regulated by an autocrine IL-6 loop, and that megakaryocytopoiesis may be independently regulated at early and late stages of differentiation.