Biomedical subjects

J E Cardier

Publications and source records attributed to J E Cardier.

10 recordsLinked to original sources

PubMed1999

Effects of megakaryocyte growth and development factor (thrombopoietin) on liver endothelial cells in vitro.

Thrombopoietin (TPO) is the major regulator of growth and differentiation of megakaryocytes. Recent studies have shown that TPO also has activity on hematopoietic lineages other than megakaryocytes. However, little is known about the effects of TPO on nonhematopoietic cells expressing the TPO receptor, such as endothelial cells (EC). We have previously shown that specific murine liver EC (LEC-1) located in the hepatic sinusoids coexpress TPO and its receptor, c-mpl. Likewise, we showed that TPO has a proliferative effect on LEC-1. In this study, we have further examined the effects of TPO on other biological functions of LEC-1. Stimulation with TPO induced secretion of proinflammatory cytokines (i.e., IL-1beta, IL-6, TNF-alpha) from LEC-1. TPO-induced proliferation of LEC-1 was synergistically enhanced with the addition of TNF-alpha. TPO also induced the proliferation of LEC-1 in the presence of IFN-gamma, which alone inhibited the growth of these cells. TPO has no effect on other endothelial cell functions such as nitric oxide production and adhesion molecule expression. These observations establish a novel activity of TPO on murine liver endothelial cells in terms of inducing cytokine production by these cells. Our results suggest that this cytokine may act synergistically with other cytokines to induce LEC-1 proliferation.

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PubMed1999

Fas (CD95, APO-1) antigen expression and function in murine liver endothelial cells: implications for the regulation of apoptosis in liver endothelial cells.

The Fas (CD95, APO-1) receptor is a membrane-associated polypeptide that can mediate apoptosis in various cell types. Although Fas receptor is expressed in endothelial cells (EC), little is known about its function in these cells. The expression of Fas by liver endothelial cells (LEC) suggests that upon stimulation, apoptosis may occur in these cells. We show that Fas is highly and constitutively expressed in cloned murine liver endothelial cells (LEC-1). In contrast, FasL expression was not detected at the protein and mRNA level in these cells. Our results show that Fas ligation in LEC-1 induces apoptotic cell death, indicating that Fas receptor is functional in these cells. The doses of Fas agonist required to induce LEC-1 apoptosis were higher than those used previously in other cells, including hepatocytes, suggesting that LEC-1 are highly resistant to the Fas apoptotic pathway. TNF treatment of LEC-1 induced up-regulation of Fas receptor on these cells. In contrast, TNF did not induce the expression of FasL on LEC-1. An increased susceptibility to Fas-mediated apoptosis was observed in TNF-treated LEC-1. Enhanced susceptibility to Fas-mediated apoptosis was also observed in LEC-1 pretreated with actinomycin D, suggesting that transcription of message coding for protective proteins is necessary to protect these cells against Fas-mediated apoptosis. Up-regulation of VCAM-1 and ICAM-1 was observed in LEC-1 treated with a dose of Fas agonist that does not induce apoptosis. To our knowledge, this is the first report that Fas mediates apoptosis in LEC, suggesting that apoptosis of these cells may participate in the liver damage observed in animals after receiving anti-Fas mAb or soluble FasL. Our findings also suggest that the Fas/FasL system may transduce activating signals independently of cell death in LEC-1.

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PubMed1998

Thrombopoietin and its receptor, c-mpl, are constitutively expressed by mouse liver endothelial cells: evidence of thrombopoietin as a growth factor for liver endothelial cells.

Present data suggest that the primary site of thrombopoietin (TPO) mRNA is the liver. Previously, we reported that specific murine liver endothelial cells (LEC-1) located in the hepatic sinusoids support in vitro megakaryocytopoiesis from murine hematopoietic stem cells suggesting that these cells may be a source of TPO. We report here that TPO and its receptor, c-mpl, are coexpressed on cloned LEC-1. Enzyme-linked immunosorbent assay (ELISA), biological assay, and flow cytometry studies confirmed the expression of both TPO and its receptor, respectively, at the protein level. TPO activity was enhanced in supernatants from LEC-1 treated with tumor necrosis factor (TNF)-alpha and gamma-interferon (INF). Our results show that TPO through its receptor stimulated the growth of LEC-1 in vitro. These observations establish LEC-1 as a novel source of TPO in the liver. To our knowledge, this is the first report that liver endothelial cells express both TPO and its receptor, c-mpl, and our findings indicate that this cytokine constitutes a growth factor for liver endothelial cells in vitro.

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PubMed1997

Extramedullary hematopoiesis in the adult mouse liver is associated with specific hepatic sinusoidal endothelial cells.

In previous work, two anatomically distinct-liver sinusoid endothelial cells (LEC): LEC-1 and LEC-2, have been described. We also reported that extramedullary hepatic hematopoiesis occurs only in close contact with LEC-1, suggesting that these cells may provide the microenvironment necessary for the maintenance and growth of hematopoietic cells. In the present work, we studied the capacity of LEC-1 and LEC-2 to maintain in vitro hematopoiesis. LEC-1 and LEC-2 were isolated and cloned from livers of adult mice. Bone marrow cells (BM) enriched with primitive hematopoietic progenitors were isolated from day-2, post-5-FU-treated mice (5-FUBMC). LEC-1 supported the maintenance and differentiation of hematopoietic progenitors for more than 6 weeks in vitro. In contrast, LEC-2 cells poorly supported the proliferation of hematopoietic cells for only two weeks of the co-culture. LEC-1 and 5-FUBMC cocultures showed cobblestone-area formation and the presence of hematopoietic progenitors that are able to form colonies (CFC) in the adhering fraction after six weeks of coculture. LEC-1 co-cultures treated with a cocktail of cytokines (stem cell factor, interleukin [IL]1alpha, IL-3, and Epo) showed that megakaryocyte (CFU-Mk) and erythrocyte progenitors (BFU-e) were present during the entire period of the culture. Granulocyte-macrophage progenitors (CFU-GM) were present only during the first three weeks of the culture. These results suggest that LEC-1, but not LEC-2, provide an appropriate hematopoietic microenvironment for supporting the proliferation and differentiation of primitive hematopoietic cells. This could explain the anatomical restriction of hematopoietic cells for growing in LEC-1 domains during liver extramedullary hematopoiesis.

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PubMed1997

Differential effect of erythropoietin and GM-CSF on megakaryocytopoiesis from primitive bone marrow cells in serum-free conditions.

In this study we have explored the effect of recombinant human erythropoietin (EPO) and recombinant murine GM-CSF on megakaryocyte progenitors (colony forming units-megakaryocyte [CFU-Mk]) using a serum-free fibrin clot assay and enriched primitive hematopoietic progenitors of marrow cells from day 4 post-5-fluorouracil-treated mice. We have monitored the production of high proliferative potential-colony forming cells ([HPP-CFC]; compact colonies, > 0.5 mm) and studied their relationship to CFU-Mk formation. EPO induced the formation of small numbers of megakaryocyte colonies, but acted together with the megakaryocyte-stimulating factors, stem cell factor (SCF) and interleukin (IL-3), to augment the size of CFU-Mk (colonies with > 50 megakaryocytes/colony). A strong correlation between the number of CFU-Mk and HPP-CFC formation from 5-fluorouracil bone marrow cells was observed when these cells were stimulated with EPO in the presence of SCF and IL-3. On the other hand, GM-CSF alone had no effect on megakaryocyte colony formation. Moreover, GM-CSF in the presence of SCF and IL-3 potentiates the HPP-CFC formation (i.e., an increase of 3.1-fold compared to the effect induced by SCF+IL-3) with strong inhibitory effects on the number and size of megakaryocyte colonies. Although several studies suggest that EPO and GM-CSF can stimulate megakaryocytopoiesis, our results indicate that neither EPO nor GM-CSF alone are sufficient to stimulate primitive progenitors committed to the megakaryocyte lineage. The fact that EPO can exert a strong effect on the size of CFU-Mk induced by SCF/IL-3 suggests that only those megakaryocyte progenitors previously stimulated by other megakaryocyte stimulating factors are able to respond to EPO. These findings may explain the physiological and clinical observations in which high levels of EPO are often associated with thrombocytosis.

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PubMed1997

IL-6 interferes with stimulation of HPP-CFC and large CFU-Mk in conjunction with cytokine combinations from primitive murine marrow cells.

The growth-promoting activities of interleukin 6 (IL-6) in combination with early-acting hematopoietic factors, i.e., stem cell factor (SCF) and interleukin-1 alpha (IL-1 alpha), on primitive hematopoietic and megakaryocyte progenitors (high proliferative potential colony-forming cells [HPP-CFC] and colony-forming units-megakaryocyte [CFU-Mk], respectively) from 5-fluorouracil (5-FU)-treated murine bone marrow cells (BMC) were evaluated in serum-free fibrin clot cultures. IL-6 in combination with SCF and IL-1 induced an irregular and abortive hematopoiesis characterized by a reduction in colony size of at least 50% over those stimulated by SCF + IL-1 + IL-3 and an inability to continue growth to day 12. Moreover, IL-6 in combination with the early-acting factors, SCF and IL-1, had no effect on the formation of HPP-CFC. IL-6 is synergistic with SCF + IL-1 on day 7 CFU-Mk but did not stimulate large day 12 CFU-Mk. Our results suggest that, in the absence of serum, IL-6 prevents the continued proliferation of early hematopoietic and megakaryocytic progenitors initiated by SCF + IL-1 + IL-3. Optimization of cytokine combinations for use in ex vivo expansion of marrow progenitors, either for stem cell transplants or gene therapy, must consider not only the number of colonies but their size, as well as the contributions of serum components.

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PubMed1997

T lymphocytes subsets in experimental iron overload.

Several abnormalities of the immune system have been reported in association with clinical and experimental iron overload. To dissect further such abnormalities, changes in lymphocyte subsets were evaluated in iron-loaded male Sprague-Dawley rats. The iron-loading protocol consisted of a total dose of irondextran (1.5 mg/Kg body weight) divided in daily intramuscular injections over twenty consecutive days. At days 0, 20, and 50 after initiation of iron injections lymphocyte subsets in blood, spleen and mesenteric lymph nodes were estimated by indirect immunofluorescence using monoclonal antibodies recognizing T cells (W3.13), the subset of helper T cells in (W3.25), and the subset of cytotoxic T cells (OX.8). By day 20, there was no change in the number of W3.25+ T cells in the blood of iron-loaded animals as compared to the controls, but the OX.8+ T cells were significantly elevated. At this time, the ratio W3.25 +/OX.8+ cells was significantly decreased (0.5 in experimental rats vs 2.0 in controls). Similar results were obtained at day 50. In the spleen, there was a decrease in the proportion of W3.25 +T cells and an increase in OX.8+ T cells at day 20. However, these values returned to normal by day 50. A negative correlation between W3.25 +/OX.8+ ratio and serum ferritin was observed in blood and spleen during iron administration. These changes were associated with abnormalities in lymphocyte proliferative response. No changes in W3.25 +/OX.8+ ratio were observed in mesenteric lymph nodes. These results demonstrate that iron overload alters the distribution of T lymphocytes in various compartments of the immune system.

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PubMed1996

Megakaryocytopoiesis in vitro: from the stem cells' perspective.

Megakaryocytopoiesis is a complex network regulated by different megakaryocyte (MK)-stimulating factors (i.e., thrombopoietin [TPO], stem cell factor [SCF], interleukin 3 [IL-3], IL-6, IL-11 and GM-CSF). Although all of these factors can affect human and murine megakaryocytopoiesis at different levels of MK development, the effect on very primitive hematopoietic stem cells (HSC) is not well understood. We have further characterized the in vitro biological activity of recombinant murine TPO, SCF and IL-3 on the maturation and proliferation of MK progenitors from different murine primitive hematopoietic cells in a fibrin clot system under serum-free conditions. Neither TPO nor SCF alone induced MK colony formation (CFU-MK) from Lin- Sca+ cells. However, isolated large and mature MKs were observed in the presence of TPO. In contrast, IL-3 exerted a potent effect on CFU-MK formation from Lin- Sca+ cells. On this population of HSC, a significant increase of large MK colonies with mature MK were obtained under those conditions in which TPO was combined with IL-3 or SCF plus IL-3. Similar results were obtained with murine bone marrow cells enriched by primitive progenitors from day 3 post-5-fluorouracil treated mice (5-FUBMC). In contrast, TPO-sensitive precursors were detected in fetal liver cells (FLC). These cells differentiate and proliferate to MK progenitors in the presence of TPO. A significant increase in the number of CFU-MK was induced when TPO was combined with either IL-3 or SCF. On these populations of primitive hematopoietic progenitors, IL-3 induced both the proliferation and differentiation of MK progenitors. Because erythropoietin and TPO share similarities between their molecules and their receptors, we studied whether these growth factors may modulate megakaryocytopoiesis from FLC. Flow cytometry analysis of FLC expressing erythroid markers demonstrated that these cells expressed c-Mpl receptor. In our in vitro studies, although EPO by itself did not induce MK colonies from FLC, it enhanced the proliferative activity of TPO. High ploidy and proplatelet-shedding MK were observed in Lin- Sca+ cells, 5-FUBMC and FLC stimulated with TPO alone or in combination with other MK-stimulating factors. Based on these observations, we propose that TPO, IL-3 and SCF constitute early MK-acting factors with differential proliferative and differentiative activities on murine stem cells. TPO by itself does not appear to be involved in the proliferation of MK progenitors from bone marrow HSC. TPO appears to induce in these cells the commitment toward MK differentiation. However, this growth factor may enhance the proliferative activity of IL-3. IL-3 is an early MK-stimulating factor able to induce in vitro the proliferation and differentiation of MK progenitors from HSC.

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PubMed1996

Thrombopoietin, a direct stimulator of viability and multilineage growth of primitive bone marrow progenitor cells.

Thrombopoietin (TPO), the ligand for c-mpl, has recently been demonstrated to be the primary regulator of megakaryocytopoiesis and platelet production. In addition, several studies have demonstrated that c-mpl is expressed on hematopoietic cell populations highly enriched in primitive progenitor cells. Here we summarize and discuss recent studies from our laboratory, as well as others, demonstrating that TPO has effects on primitive hematopoietic progenitor cells. When acting alone, TPO stimulates little or no growth, but promotes viability and suppresses apoptosis of murine multipotent (Lin- Sca-1+) bone marrow progenitor cells in vitro. In addition, TPO directly and potently synergizes with other early acting cytokines (kit ligand, flt3 ligand and interleukin 3) to promote multilineage growth of the same progenitor cell population. Although it remains to be established whether TPO also acts on the long-term reconstituting pluripotent stem cells, these studies combined with progenitor cell studies in c-mpl-deficient mice, suggest that TPO, in addition to its key role in platelet production, might also have an important impact on early hematopoiesis.

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PubMed1995

Lipid peroxidation and changes in T lymphocyte subsets and lymphocyte proliferative response in experimental iron overload.

This study was undertaken to investigate the hypothesis that lipid peroxidation might be associated with immunological abnormalities in experimental hemosiderosis. The correlation between the degree of plasma and spleen lipid peroxidation with lymphocyte proliferative response and with the proportion of T lymphocyte subsets was studied in normal and iron overloaded male Sprague Dawley rats. The iron-loading protocol consisted of a total dose of iron-dextran (1.5 mg/Kg body weight) divided in daily i.m. injections over twenty consecutive days. Lipid peroxidation was measured by the thiobarbituric acid assay in plasma and in homogenates of spleen. Plasma lipid peroxide level increased rapidly after i.m. administration of iron-dextran and decreased sharply at 48 h after the last injection. Conversely, a progressive increase of lipid peroxidation in homogenates of spleen was observed in the course of the iron overload protocol, remaining high even at 50 days after initiation of iron-dextran injections. The increase of spleen lipid peroxide levels was associated with decreased lymphocyte proliferative response to Con A in iron overloaded rats. The addition of superoxide dismutase and catalase to lymphocyte cultures reversed the inhibition of the proliferative response, implicating reactive species of oxygen as the causative agents of these alterations. These effects may be related with the enhanced membrane and DNA damage occurring during intracellular and extracellular peroxidation. Negative correlations between helper/cytotoxic ratio and malondialdehyde levels were obtained in blood and spleen during iron administration. These results supports the hypothesis that lipid peroxidation plays a role in the immunological abnormalities observed in experimental hemosiderosis.

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