Anne Van der Meeren

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

PURPOSEnThe 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).nnnMETHODS AND MATERIALSnHematopoietic 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.nnnRESULTSnTPO 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.nnnCONCLUSIONnThese results demonstrate that the multilineage hematopoietic effects of TPO may be advantageously used to protect against lethal bone marrow failure following high dose TBI.

Inflammatory Reaction and Changes in Expression of Coagulation Proteins on Lung Endothelial Cells after Total-Body Irradiation in Mice

Abstract Van der Meeren, A., Vandamme, M., Squiban, C., Gaugler, M-H. and Mouthon, M-A. Inflammatory Reaction and Changes in Expression of Coagulation Proteins on Lung Endothelial Cells after Total-Body Irradiation in Mice. Radiat. Res. 160, 637–646 (2003). Inflammatory reaction is a classical feature of radiation exposure, and pneumonitis is a dose-limiting complication in the handling of hematological disorders treated with total-body irradiation. In the present study, we first evaluated the inflammatory response in C57BL6/J mice exposed to lethal doses of γ rays treated with antibiotics or not. Both interleukin 6 and KC (also known as Gro1) were increased in the plasma 10 to 18 days after radiation exposure, independent of bacterial infection, whereas fibrinogen release was linked to a bacterial infection. Furthermore, both Il6 and KC were increased in the lungs of irradiated mice. Our second objective was to characterize the endothelial cell changes in the lungs of total-body-irradiated mice. For this purpose, a quantitative RT-PCR was used to determine the expression of genes involved in inflammatory and coagulation processes. We found that the adhesion molecules P-selectin and platelet endothelial cell adhesion molecule 1 were up-regulated, whereas E-selectin remained unchanged. Tissue factor expression was up-regulated as well, and thrombomodulin gene expression was down-regulated. The investigation by immunohistochemistry of adhesion molecules confirmed the increase in the basal expression of both P-selectin and platelet endothelial cell adhesion molecule 1 on pulmonary endothelial cells. All together, our results suggest the involvement of endothelial cells in the development of radiation-induced inflammatory and thrombotic processes.

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 10u2003Gy 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 (5–15u2003Gy) 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 (1,2u2003Gy) 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.

Administration of Recombinant Human IL11 after Supralethal Radiation Exposure Promotes Survival in Mice: Interactive Effect with Thrombopoietin

Abstract Van der Meeren, A., Mouthon, M-A., Gaugler, M-H., Vandamme, M. and Gourmelon, P. Administration of Recombinant Human IL11 after Supralethal Radiation Exposure Promotes Survival in Mice: Interactive Effect with Thrombopoietin. Radiat. Res. 157, 642–649 (2002). In the present study, we evaluated the therapeutic potential of recombinant human IL11 in lethally irradiated C57BL6/J mice exposed to γ rays. IL11 administered for 5 consecutive days beginning 2 h after total-body irradiation with 8 or 9 Gy 60Co γ rays resulted in a significant increase in 30-day survival. When IL11 was administered, only a slight improvement in the hematopoietic status (both blood cell counts and progenitor cells) was observed after an 8-Gy exposure, and no improvement in hematopoietic reconstitution was observed after 9 Gy total-body irradiation. The enhancement of fibrinogen in the plasma of irradiated animals suggested the importance of infections in the death of animals. IL11 was able to limit the increase in fibrinogen levels. However, prevention of bacterial infections by antibiotic treatment, although it delayed death, was ineffective in promoting survival either in placebo-treated and IL11-treated mice. IL11 was administered along with thrombopoietin (TPO) or bone marrow transplantation to limit the hematopoietic syndrome, in addition to antibiotic treatment. When IL11 was combined with TPO, a potent stimulator of hematopoiesis, the survival of animals which had been irradiated with 10 Gy 137Cs γ rays was increased significantly compared to those treated with IL11 or TPO alone. Furthermore, an interactive effect of TPO and IL11 on hematopoietic reconstitution was observed. Similarly, IL11 in combination with bone marrow transplantation enhanced survival after 15 Gy 137Cs γ rays. These data suggest that the effect of IL11 on the hematopoietic system is only moderate when it is used alone in supralethally irradiated mice but that the effect is improved in the presence of a hematopoietic growth factor or bone marrow transplantation.

Characterization of the acute inflammatory response after irradiation in mice and its regulation by interleukin 4 (Il4).

Abstract Van der Meeren, A., Monti, P., Lebaron-Jacobs, L., Marquette, C. and Gourmelon, P. Characterization of the Acute Inflammatory Response after Irradiation in Mice and its Regulation by Interleukin 4 (Il4). Radiat. Res. 155, 856–863 (2001). The aim of this study was to determine the effects of total-body irradiation of mice on the acute release of a panel of several mediators of inflammation and to evaluate the efficacy of Il4 in regulating these radiation-induced modifications. We studied the effects of exposure of C57BL6/J mice to 8 Gy γ rays on the early release of cytokines, chemokines, acute-phase proteins, prostaglandins and corticosterone in either plasma or tissues compared to those observed after intraperitoneal injection of lipopolysaccharide from 1 h to 3 days after stimulation. During the characterization of the acute inflammatory response induced by radiation or lipopolysaccharide, we observed differences both in the type of mediators produced and in the time course of release. We next determined the anti-inflammatory potential of Il4 in this model of total-body irradiation. We found that Il4 was able to down-regulate the radiation-induced production of mediators of inflammation such as Gro1 (also known as KC, N51) in plasma and lung, corticosterone in blood, Il1b in lung, and prostaglandin E2 in colon, suggesting the anti-inflammatory potential of Il4 in regulating the radiation-induced response.

Irradiation increases the interactions of platelets with the endothelium in vivo: Analysis by intravital microscopy

Abstract Mouthon, M-A., Vereycken-Holler, V., Van der Meeren, A. and Gaugler, M-H. Irradiation Increases the Interactions of Platelets with the Endothelium In Vivo: Analysis by Intravital Microscopy. Radiat. Res. 160, 593–599 (2003). Adhesion of platelets to the endothelium is believed to be a major factor contributing to thrombosis and vascular occlusion after radiotherapy or endovascular irradiation. In the present study, platelet–endothelium interactions were analyzed in vivo by intravital microscopy in mesenteric venules of mice according to three parameters: (1) platelet rolling, (2) platelet adhesion, and (3) the presence of platelet clusters. A 10-Gy total-body irradiation of mice resulted in an increase in the frequency of appearance of these three types of platelet–endothelium interactions in postcapillary venules 6 and 24 h after exposure, whereas only minor alterations were seen in large venules. In addition, the duration of platelet adhesion was increased 24 h after irradiation in both postcapillary and large venules. However, P-selectin was not up-regulated on the platelet membrane and platelet-leukocytes were not seen rolling together, suggesting that changes in platelet–endothelial cell interaction result from endothelial cell activation rather than platelet activation. Our data suggest that irradiation transforms resting endothelial cells to a pro-adhesive surface for platelets, which could ultimately lead to thrombosis.

Combinations of Cytokines Promote Survival of Mice and Limit Acute Radiation Damage in Concert with Amelioration of Vascular Damage

Abstract Van der Meeren, A., Mouthon, M-A., Vandamme, M., Squiban, C. and Aigueperse, J. Combinations of Cytokines Promote Survival of Mice and Limit Acute Radiation Damage in Concert with Amelioration of Vascular Damage. Radiat. Res. 161, 549–559 (2004). Recovery from hematopoietic aplasia is a predominant factor in the survival of total-body-irradiated mice within 30 days after exposure. However, other radiation-induced pathophysiological events have been shown to play a role, among which an inflammatory reaction must be considered. In the present study, we evaluated the therapeutic potential of a hematopoietic growth factor (thrombopoietin, Tpo) and pleiotropic cytokines (Il4 or Il11), used alone or in combination, on the survival of mice, hematopoietic reconstitution, inflammatory reaction and vascular changes. All treatments including Tpo induced a higher level of survival than did treatment with a placebo, with combinations being the most efficient. The increased survival could not be explained solely by an improved hematopoietic recovery. Treatments with Tpo also reduced the level of the chemokine KC in plasma and the level of expression of mRNA for inflammatory and coagulation proteins in the lungs of irradiated mice. In addition, radiation- induced vascular hyperpermeability was reduced with the use of Tpo. In summary, our results show that Tpo may improve survival by limiting vascular leakage, which in turn could limit inflammatory reactions and the ensuing tissue damage.

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 Decorporation of Americium by Pulmonary Administration of DTPA Dry Powder after Inhalation of Aged PuO2 Containing Americium in Rats

Abstract After inhalation of plutonium oxides containing various percentages of americium in rats, we identified an acellular transient pulmonary compartment, the epithelial lining fluid (ELF), in which a fraction of actinide oxides dissolve prior to absorption and subsequent extrapulmonary deposit. Chelation therapy is usually considered to be poorly efficient after inhalation of actinide oxides. However, in the present study, prompt pulmonary administration of diethylenetraminepentaacetic acid (DTPA) as a dry powder led to a decrease in actinide content in ELF together with a limitation of bone and liver deposits. Because americium is more soluble than plutonium, higher amounts of americium were found in ELF, extrapulmonary tissues and urine. Our results also demonstrated that the higher efficacy of DTPA on americium compared to plutonium in ELF induced a preferential inhibition of extrapulmonary deposit and a greater urinary excretion of americium compared to plutonium. All together, our data justify the use of an early and local DTPA treatment after inhalation of plutonium oxide aerosols in which americium can be in high proportion such as in aged compounds.