PECAM-1 (CD31) is required for interactions of platelets with endothelial cells after irradiation.

Sustained adhesion of platelets to endothelial cells (EC) is believed to contribute to thrombosis and vascular occlusions following radiation exposure leading to organ functional impairment and even death. Our objective was to evaluate the role of platelet endothelial cell adhesion molecule (PECAM)-1 in the prothrombotic response of EC after irradiation. Endothelial PECAM-1 expression was determined by cell-enzyme linked immunosorbent assay (ELISA) on human microvascular EC from lung (HMVEC-L) up to 21 days after a 10 Gy irradiation. Platelet- and leukocyte-endothelial cell interactions were assessed using a flow adhesion assay with fluorescently labeled whole blood, and the function of PECAM-1 in these processes was measured by using blocking antibody. PECAM-1 expression was significantly increased on irradiated HMVEC-L and remained elevated at 21 days. Anti-PECAM-1 antibody significantly inhibited adhesion of single platelets and thrombi on irradiated HMVEC-L. This inhibitory effect persisted at day 21. Anti-PECAM-1 also reduced leukocyte adhesion to irradiated HMVEC-L. The up-regulation of endothelial PECAM-1 following radiation exposure is persistent. PECAM-1 plays a key role platelet adhesion/aggregation on irradiated EC. Therefore, strategies targeting this adhesion molecule may prevent the development of radiation pathologies.

Radiation effects on circulating and endothelial cell interactions studied by quantitative real-time videomicroscopy.

PURPOSE: To quantify in vitro the functional consequences of irradiation on the interactions between leukocytes or platelets and endothelial cells (EC) in flowing whole blood using a parallel-plate flow chamber and real-time videomicroscopy. MATERIALS AND METHODS: The parallel-plate flow chamber was calibrated to determine the dynamic parameters of the flow channel. Fluorescent-labelled whole blood was perfused at wall shear rates of 25, 75 and 500 s(-1) over a monolayer of human microvascular EC-lung (HMVEC-L) with or without irradiation at 10 Gy. The adhesion of leukocytes and platelets on EC was quantified by videomicroscopy and image analysis. RESULTS: Calibration of the parallel-plate flow chamber showed that flow in the chamber was laminar and steady and had a parabolic velocity profile, thus simulating physiological flow conditions. Flow assay revealed that rolling, mean rolling velocity and firm adhesion of leukocytes was increased following irradiation of EC. Irradiation also favoured platelet adhesion to EC. CONCLUSIONS: The results of an in vitro flow assay with whole blood showed that under physiological flow conditions, irradiation affected the function of EC; pro-inflammatory and thrombogenic responses were enhanced, which may contribute to in vivo radiation-induced vascular occlusion and fibrosis.

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.

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.

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.

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.

Differential regulation by IL-4 and IL-10 of radiation-induced IL-6 and IL-8 production and ICAM-1 expression by human endothelial cells.

Radiation exposure results in an inflammatory reaction with acute as well as subacute consequences. Leukocyte infiltration is one of the predominant early histological changes and involves both cytokines and adhesion molecules. Endothelial cells play a key role in this reaction. We have previously shown the increased production of interleukin 6 (IL-6) and IL-8 and the upregulation in intercellular adhesion molecule 1 (ICAM-1) expression by HUVEC following gamma ray exposure. In the present study, we used the cytokines IL-4 and IL-10 to regulate these radiation-induced manifestations. Human umbilical vascular endothelial cells (HUVEC) were treated with IL-4 and IL-10 (50 pg/ml) either before or after 10- Gy irradiation. Three and seven days after irradiation, IL-6 and IL-8 production by HUVEC (either treated or non-treated) was assessed by enzyme-linked immunosorbent assay (ELISA). Our results show that IL-4, when added after irradiation, reversed the radiation-induced increase in IL-8 production, although slightly increased IL-6 production. IL-10 decreased both IL-8 and IL-6 production when added after irradiation. ICAM-1 expression was evaluated 3 days after irradiation by flow cytometry. The radiation-induced upregulation in ICAM-1 expression remained unaffected by the use of IL-4. Altogether, our results show that radiation-induced endothelial cell activation may be ameliorated by IL-4 and/or IL-10, which is of significance in designing strategies for cytokine-mediated intervention and/or therapy of radiation damage.

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.

Characterization of the response of human bone marrow endothelial cells to in vitro irradiation.

Endothelial cell dysfunction is a classic consequence of radiation damage. Bone marrow endothelial cells (BMEC) are a critical component of the stroma in the regulation of haemopoiesis. In animal models, radiation-induced injury of BMEC has been described and a role for BMEC in haemopoietic regeneration after irradiation has been suggested. However, functions of BMEC involved in the haemopoietic regeneration have not been assessed. Therefore we studied the functional response of human BMEC to irradiation using the transformed human BMEC line (TrHBMEC) irradiated with 2. 5 or 10Gy. Our results showed a time- and a dose-dependent increase in damage to irradiated TrHBMEC measured by a decreased number of adherent cells which correlated with increased apoptosis and augmented release of soluble ICAM-1 and von Willebrand factor. 2 Gy irradiated TrHBMEC expressed more ICAM-1 on their surface than non-irradiated cells, whereas no change in VCAM-1, E-selectin and PECAM-1 expression was observed. An increased production of G-CSF, GM-CSF, IL-8, IL-6, IL-1alpha, IL-11, MIP-1alpha and SCF and no production of LIF, TNF-alpha, TPO and IL-3 by 2 Gy irradiated TrHBMEC was observed. The haemopoietic supportive function of TrHBMEC was not altered after a 2 Gy exposure. These results suggest that although radiation induces endothelial cell damage, irradiated cells still support the proliferation and the differentiation of CD34+ haemopoietic cells.

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.

Ionizing radiation enhances IL-6 and IL-8 production by human endothelial cells.

Irradiation exposure is known to induce an inflammatory reaction. Endothelial cells play a crucial role both in the inflammatory process and in radiation damage. Therefore, supernatants and cell lysates of (60)Co-irradiated human umbilical vein endothelial cells (HUVEC) have been assessed for the presence of pro-inflammatory cytokines. After gamma irradiation, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha remained undetectable in both cell supernatants and cell lysates. However, a dose-dependent increase in the production of IL-6 and IL-8 has been demonstrated up to 6 days after exposure. These data indicate that the pro-inflammatory cytokines IL-6 and IL-8 may be involved in the inflammatory response of vascular endothelium induced by exposure to ionizing radiation.

Expression and function of integrins on hematopoietic progenitor cells.

The growth and differentiation of hematopoietic stem cells are highly dependent on regulatory molecules produced by stromal cells of the marrow environment. Evidence has accumulated over the past years which shows that adhesive receptors on hematopoietic cells and their ligands on stromal cells and extracellular matrix play a crucial role in these interactions. Integrins of the beta 1 family, mostly VLA-4 and VLA-5, are the best characterized and have been identified on committed progenitor cells of the hematopoietic hierarchy as well as on more primitive stem cells defined by their long-term repopulating capacity assayed in vitro as well as in vivo. Functional assays demonstrate that most progenitor cells efficiently bind to ECM components through beta 1 integrins and lineage- and maturation stage-specific differences have been described. Evidence exists on the direct control of late erythroid differentiation by VLA-4, but whether or not the triggering of beta 1 integrins is critically required for hematopoietic stem cell functioning at more immature steps is unclear. Many other integrin and non-integrin receptors involved in adhesive interactions are expressed on hematopoietic progenitor cells and tightly regulated during differentiation but their function is still controversial. Our main purpose in this review is to describe recent advances in the knowledge of integrin expression on hematopoietic progenitor cells in both mouse and man. The emerging importance of the synergy between integrins and cytokine signalling pathways in the regulation of hematopoietic differentiation will also be discussed.

Coexpression of two fibronectin receptors, VLA-4 and VLA-5, by immature human erythroblastic precursor cells.

Human erythroblastic precursor cells adhere to fibronectin (Fn) but the exact nature of the receptors mediating this interaction has not been characterized. In this study, we report data showing that immature human erythroblasts express the integrins VLA-4 and VLA-5 and that both these molecules act as fibronectin receptors on these cells. We have recently demonstrated that adhesion to Fn of purified human CFU-E and their immediate progeny preproerythroblasts was inhibited by antibodies directed against the human fibronectin receptor (VLA-5). Here we have extended those results and characterized by immunoprecipitation with specific antibodies the integrins expressed on surface-labeled normal human immature erythroblasts. A polyclonal antibody recognizing the common VLA beta 1 subunit yielded two polypeptides of 120 and 160 kD. Our data further demonstrate that the polypeptide of 160 kD contains alpha subunits corresponding to both alpha 4 and alpha 5. Thus, erythroblast lysates prepared in 0.3% CHAPS and immunoprecipitated with antibodies which specifically recognize the alpha 4 subunit showed a heterodimer with peptides of 120 (beta 1) and 160 kD (alpha 4) and the additional peptides of 70 and 80 kD which usually coprecipitate with the alpha 4 chain. On the other hand, specific anti-alpha 5 antibodies immunoprecipitated an alpha 5/beta 1 complex with peptides of 120 and 160 kD which under reducing conditions migrated as a single band of 130 kD. Similar experiments performed with an erythroleukemic cell line (KU 812) showed that these cells also coexpress both the VLA-4 and VLA-5 members of the integrin family. Furthermore, monoclonal antibodies recognizing the VLA alpha 4 chain blocked the adhesion of immature erythroblasts to Fn-coated surfaces, thus demonstrating that, as VLA-5, VLA-4 is also a functional Fn receptor on these cells.

Association between leukemic erythroid progenitors and bone marrow macrophages.

Previous ultrastructural investigations have shown that the erythroblastic island is composed of erythroblasts at different stages of maturation which are intimately associated with a central macrophage. However, it is still unclear at which stage of erythroid differentiation this interaction occurs, mainly because of the lack of purified populations of normal erythroid progenitors [erythroid colony-forming units (CFU-E) and erythroid burst-forming units (BFU-E)] and early precursor cells (proerythroblasts) and because of our limited knowledge of their ultrastructural characteristics. In the present work we analyzed the ultrastructure of CFU-E enriched from normal human bone marrow by avidin-biotin immune rosetting and leukemic blasts of erythroid origin from two patients. Normal and leukemic CFU-Es were defined as glycophorin A (GPA)-negative blasts, devoid of rhopheocytosis, containing some ferritin molecules, either free in the cytoplasm or associated with theta-granules (theta-Gr) in the Golgi zone. Peroxidase activity was detected in the endoplasmic reticulum of these blasts. A preproerythroblast stage was identified, which corresponded to an intermediate phenotype with few GPA sites and rhopheocytosis. In contrast to hemoglobin synthesis, which was absolutely dependent on the presence of erythropoietin (Epo) during culture for 24 hours, ferritin molecules accumulated in the absence of Epo. Interestingly, leukemic CFU-E-like blasts were always in contact with bone marrow macrophages and adhesion between these cell types resisted mechanical dissociation. This result suggests that erythroid progenitors may be part of the erythroblastic island. The mechanisms involved in erythroblast-macrophage binding are still unknown, but the expression by macrophages and erythroid progenitors of receptors for fibronectin and thrombospondin (TSP), as well as their respective ligands in the case of macrophages, suggests that these molecules could be involved in the formation of the erythroblastic island.

Adhesive properties of human erythroblastic precursor cells.

Interactions between hematopoietic precursor cells and their surrounding marrow environment are essential for hematopoietic differentiation. These occur in part through the production of regulatory molecules by marrow stromal cells and their local concentration by components of the extracellular matrix (ECM), but direct cell-cell or cell-matrix contacts are likely to also play an essential role. During the past several years, we have characterized the adhesive properties of human hematopoietic precursor cells on two substrates, marrow fibroblastic ECM and purified plasma fibronectin (Fn). We have shown that adhesion to marrow ECM and Fn was a selective property of erythroblastic cells and was expressed by normal erythroblastic progenitors (CFU-E and BFU-E), as well as by erythroleukemic cell lines (K 562), but only by a small proportion of CFU-GM. Furthermore, attachment to Fn was very precisely regulated during erythroblastic differentiation as shown by the loss of Fn-adhesion in reticulocytes derived from purified immature erythroblasts induced to differentiate in vitro. The physiological relevance of these results is not well understood, but we speculate that adhesion to ECM components helps stem cells to locate in a favorable environment, and that loss of this property in reticulocytes is required before they cross the marrow-blood barrier.

Loss of attachment to fibronectin with terminal human erythroid differentiation.

Human erythroblastic progenitors (colony-forming unit-erythroid [CFU-E] and burst-forming unit-erythroid [BFU-E]) have been shown to attach to fibronectin (Fn), a property that might be involved in the local regulation of erythropoiesis. In this study, we have investigated changes in cell attachment to Fn upon terminal erythroid differentiation. We first purified CFU-E from human marrow by avidin-biotin immune rosetting. This negative selection procedure yielded a cell population containing approximately 80% blasts that, after characterization by colony-assays and electron microscopy, appeared to consist of CFU-E (10% to 15%) and their immediate progeny (85% to 90%), here defined as "preproerythroblasts." In the presence of erythropoietin, purified cells differentiated into reticulocytes in 7 to 10 days. Cell attachment to Fn was inversely correlated to the stage of differentiation of the erythroid cell: more than 50% of the CFU-E population reproducibly adhered to Fn, whereas at most 30% of the preproerythroblasts had the same capacity. Adhesion was further lost at late maturation stages, and a constant finding was the inability of reticulocytes to adhere to Fn. Finally, CFU-E adhesion to Fn was blocked by polyclonal lgG raised against the Fn receptor and by a monoclonal antibody against VLA-5. These results demonstrate that adhesion to Fn is developmentally regulated during normal human erythropoiesis. Restriction of its expression to CFU-E and its first divisions strikingly correlates with the migratory capacity of these cells.