The Sertoli cell-germ cell interactions and the seminiferous tubule interleukin-1 and interleukin-6 system.
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Biomedical subjects
C Piquet-Pellorce
Publications and source records attributed to C Piquet-Pellorce.
Leukemia inhibitory factor expression and regulation within the testis.
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine known to control the proliferation and survival of stem cells including primordial germ cells and gonocytes. This led us to study the origin and regulation of testicular LIF. The LIF transcript was detected in the rat testis by RT-PCR from 13.5 days postcoitum until adulthood. LIF expression was investigated further in vitro in seven different highly purified testicular cell populations using RT-PCR and bioassays combined with neutralization experiments. LIF was found to be produced by peritubular cells and, to a much lesser extent, by the other testicular somatic cell types. No LIF was detected in meiotic and postmeiotic germ cell-conditioned medium, and only low levels of LIF were detected in spermatogonia-conditioned medium. Large amounts of bioactive LIF were measured in testicular lymph. While LIF production was greatly enhanced in presence of serum, lipopolysaccharide, and TNFalpha further increased this production in peritubular and Sertoli cells, and human CG enhanced Leydig cell LIF production. In conclusion, peritubular cells are the principal source of testicular LIF, probably accounting for its high concentration in the lymph. Given the proliferative effect of LIF on immature germ cells, we suggest that peritubular LIF plays an important role in the regulation of testicular function.
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Are LIF and related cytokines functionally equivalent?
Leukemia inhibitory factor (LIF) is structurally related to interleukin-6 (IL-6), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF). Since LIF-deficient mice do not exhibit overt phenotypic effects in cell types known to be targets for LIF action in vitro, we examined the ability of IL-6, OSM, and CNTF to reproduce the effects of LIF in five different bioassays. OSM, CNTF, and LIF are able to promote embryonic stem cell growth and to maintain them in an undifferentiated state as marked by a high alkaline phosphatase activity (ED50 are, respectively, 0.5, 3 and 1 ng/ml). Whereas LIF and OSM maintain close to 100% of ES cells in an undifferentiated state, CNTF, at optimal concentrations, prevents differentiation of only 60% of the ES population. Murine 7TD1 hybridoma cell growth is induced only in the presence of IL-6 (ED50 = 0.1 ng/ml). Both LIF and OSM stimulate DA1a cell proliferation (ED50 are, respectively, 1 and 12 ng/ml). OSM appears, therefore, to act as a weak agonist of LIF-dependent processes on murine cells, however, with a 10-fold lower specific activity than that of LIF, which is in agreement with human OSM cross-reacting with the murine LIF-R. Though IL-6, LIF, and OSM all stimulate haptoglobin and fibrinogen production by human HepG2 hepatoma cells, the dose-response curves of these three factors exhibit very different characteristics. CNTF stimulates acute-phase protein production by HepG2 cells only at high concentrations (greater than 1 microgram/ml). A549 epithelial cells are subjected to growth inhibition only in the presence of OSM (ED50 = 6 ng/ml), even though they expressed LIF-R and gp130 transcripts. These data suggest that OSM and LIF act on human cells through different receptors. Altogether, these results indicate that none of the factors examined in this study are precisely interchangeable in terms of their biological actions.
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Characterization of a binding protein for leukemia inhibitory factor localized in extracellular matrix.
Leukemia Inhibitory Factor (LIF) interacts with two classes of high affinity binding sites on rat UMR cells cultured in monolayer. One class of binding sites was found to be localized in the extracellular matrix (ECM) after removal of cells from the culture dish. The interaction of LIF with ECM-localized binding sites is not dependent upon either glycosylation of LIF or the presence of extracellular glycosyaminoglycans. Chemical cross-linking studies demonstrate that LIF interacts with a 200-kD cell-associated protein and a 140-kD ECM-localized protein. A 140-kD protein could also be specifically precipitated from solubilised metabolically radiolabeled UMR ECM by antibodies directed against LIF by virtue of its ability to form a stable complex with unlabeled LIF. In addition, soluble LIF associated with this ECM-localized protein is biologically active in terms of inhibition of ES cell differentiation. The properties of ECM-localized 140-kD species are very similar to those of the secreted form of the LIF receptor suggesting that the ECM localization of LIF and LIF signal transduction may be closely coupled.
Alkaline Phosphatase↗
GM-CSF in association with IL-1 triggers day-8 CFU-S into cell cycle: role of histamine.
Our recent evidence for the requirement of endogeneous histamine in IL-3-induced proliferation of day-8 CFU-S has prompted us to investigate whether or not GM-CSF, which shares with IL-3 the ability to stimulate bone marrow histamine synthesis, could also affect the cell cycle status of CFU-S via this mediator. We show herein that recombinant GM-CSF alone fails to trigger day-8 CFU-S into S phase, but supports their survival. However, in the same experimental conditions, GM-CSF in combination with IL-1 induces a CFU-S proliferation similar to that obtained in response to IL-3, while IL-1 by itself has no effect on this biological activity. We further provide evidence that this phenomenon is completely abolished: i) by preventing GM-CSF-induced histamine synthesis by alpha-FMH, the specific inhibitor of histidine decarboxylase (HDC), or ii) by blocking the binding sites of H2 histamine receptors with their specific antagonist oxmetidine. Similar results are obtained when progenitor-enriched bone marrow cells are used instead of the unfractionated population. In addition, we provide an argument in support of a histamine receptor modulation by GM-CSF that could explain the lack of effect of factor-induced histamine on day-8 CFU-S cell cycling. Indeed, the entry of these progenitors into S phase that is normally promoted by dimaprit, a specific histamine H2 receptor agonist, is abolished by a preincubation with GM-CSF. Taken together, our data support the conclusion that IL-1 makes CFU-S sensitive to GM-CSF-induced endogeneous histamine that will trigger them into cell cycle, while GM-CSF alone has no such effect on this biological activity.
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Effect of lipopolysaccharides on histamine synthesis by hematopoietic cells.
We show herein that lipopolysaccharides (LPS), in vitro, synergize with GM-CSF to increase histamine synthesis by murine bone marrow cells. LPS has no effect on its own and does not potentiate histamine synthesis promoted by IL-3, the only other cytokine sharing this biological activity with GM-CSF. Despite the fact that GM-CSF and LPS synergistically increase PGE2 levels, the potentiating effect of LPS does not require PGE2 that have been previously shown to enhance GM-CSF-induced histamine synthesis. We provide evidence that this effect of LPS on histamine production by bone marrow cells is mediated by the intracellular cAMP transduction signal. In addition, LPS and cAMP enhance GM-CSF-induced histidine decarboxylase activity, showing that both substances act on histamine synthesis. Contrary to in vitro results, LPS injection into mice induces an increase in both intracellular histamine and HDC activity in bone marrow cells. Our results support the conclusion that this effect is mediated by GM-CSF. In conclusion, LPS appears to be a powerful HDC inducer in hematopoietic organs because of its ability, on one hand, to induce circulating GM-CSF and, on the other hand, to potentiate GM-CSF induction of HDC.
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Prostaglandin E2 potentiates granulocyte-macrophage colony stimulating factor-induced histamine synthesis in bone marrow cells: role of cAMP.
Histamine synthesis in response to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) by murine hematopoietic cells is strikingly potentiated by prostaglandin E2 (PGE2). This synergy is mediated by an increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP), since: (a) exogeneous and endogeneous cAMP generated either by forskolin or IBMX potentiate GM-CSF-induced histamine synthesis, (b) the maximal potentiating effects of PGE2 and cAMP are not cumulative, and (c) GM-CSF together with PGE2 enhances intracellular cAMP content in a bone marrow population enriched for GM-CSF target cells. cAMP and PGE2 enhance histidine decarboxylase activity induced by GM-CSF showing that both factors act on histamine synthesis rather than on its release. Conversely, histamine synthesis promoted by Interleukin 3 (IL-3), the unique cytokine sharing this property with GM-CSF, is not modulated by PGE2 or cAMP, suggesting two distinct mechanisms for the induction of this biological activity in hematopoietic cells.
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Antigenic challenge of immunized mice induces endogeneous production of IL-3 that increases histamine synthesis in hematopoietic organs.
Antigenic challenge of Nippostrongylus brasiliensis-infected mice induces a striking increase in histidine decarboxylase (HDC) activity in both spleen and bone marrow cells. This enhancement takes place within 1 h after injection, with a maximum at 4 h and a return to pretreatment values 20 h later. It is associated with the appearance of IL-3 in the sera of these mice. In addition, the intracellular histamine content in both hematopoietic organs is concomitantly increased. A similar injection of worm Ag into normal mice has no significant effect. Comparable enhancement of HDC activity and intracellular histamine content with almost identical kinetics is promoted by i.v. injection of rIL-3 into normal mice. Moreover, HDC levels in infected mice are increased to the same extent in response to either specific antigen or rIL-3 injection. Taken together these results support the conclusion that antigenic challenge of immunized mice induces endogeneous IL-3 which, in turn, promotes a rapid increase in histamine synthesis in hematopoietic organs.
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New role for histamine in interleukin-3-induced proliferation of hematopoietic stem cells.
This study investigated the effect of histamine generated by murine bone marrow cells in response to IL-3 on one particular biological activity of this growth factor, i.e., triggering of cells forming colonies in spleen (CFU-S) into S phase. Evidence is provided that i) IL-3-induced day-8 CFU-S cell cycling, evaluated by hydroxy-urea suicide, is completely abrogated when the binding of histamine to its H2 receptors is blocked by the specific antagonist oxmetidine, whereas cetirizine, a H1 receptor antagonist, is ineffective; and ii) the entry of day-8 CFU-S into S phase in response to IL-3 is likewise abolished when the histamine synthesis promoted by the growth factor is prevented by alpha-fluoromethylhistidine, a specific inhibitor of the histamine-forming enzyme, histidine decarboxylase. Similar results are obtained with both drugs, when a progenitor-enriched bone marrow population is used instead of total cells. Furthermore, i.v. injection of recombinant (r)IL-3 results within 2 hr in a substantial increase in bone marrow cell histamine synthesis together with triggering of day-8 CFU-S into cycle, the latter being completely abolished by a simultaneous injection of the H2 histamine receptor antagonist oxmetidine. Thus, our findings support the notion that both in vitro and in vivo the proliferation of early CFU-S in response to IL-3 is modulated by histamine via its H2 receptors. This conclusion is also consistent with the observation that dimaprit, a specific agonist of these receptors not only enhances the sensitivity of day-8 CFU-S to HU after a 2 hr incubation with bone marrow cells but also increases, to the same extent as IL-3, the number of colonies formed in irradiated spleens after a 5 hr pretreatment.
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Interleukin 1 and/or tumor necrosis factor-alpha synergize with granulocyte-macrophage colony-stimulating factor to enhance histamine synthesis in hematopoietic cells: role of prostaglandin E2.
Previously, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates histamine synthesis by normal murine hematopoietic cells. Addition of either interleukin (IL) 1 (alpha or beta) or murine recombinant tumor necrosis factor (TNF)-alpha to murine recombinant GM-CSF (at optimal or suboptimal concentrations) enhances its activity on bone marrow histamine synthesis up to 70%. Evidence is provided that these synergies between GM-CSF and IL 1 or TNF-alpha are mediated by prostaglandin E2 (PGE2) production since (a) GM-CSF together with either IL 1 or TNF-alpha stimulates PGE2 synthesis by bone marrow cells, while none of these factors does it alone; (b) exogenous PGE2 (ranging from 10(-6) M to 10(-10) M) potentiates GM-CSF-induced histamine synthesis in a dose-dependent manner; and (c) indomethacin, a cyclooxygenase inhibitor, completely abrogates the synergistic action of IL 1 and TNF-alpha on GM-CSF-induced histamine generation. Conversely, histamine synthesis promoted by IL 3, the unique cytokine sharing this property with GM-CSF, cannot be modulated by IL 1, TNF-alpha or PGE2, suggesting two distinct mechanisms for the induction of this biological activity in hematopoietic progenitor cells.
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[Demonstration of synergy between the factor stimulating granulocytes and macrophages colonies and interleukin-I for the stimulation of prostaglandin E2 synthesis by bone marrow cells].
Conditioned medium from P388 D1 cell line containing interleukin 1 (IL-1) and granulocyte macrophage colony stimulating factor (GM-CSF) can stimulate prostaglandin E2 (PGE2) production by murine bone marrow cells. In this work, we show that although GM-CSF (either purified from P388 D1 CM or murine recombinant GM-CSF) does not significantly alter bone marrow cell PGE2 production, its presence in P388 D1 CM is however necessary to induce this effect since the presence of anti GM-CSF antiserum completely abrogated the increase in PGE2 production in response to P388 D1 CM. In addition IL-1 tested alone does not not modify PGE2 release by bone marrow cells. However, the simultaneous addition of IL-1 and GM-CSF markedly increases PGE2 production. Thus, the ability of P388 D1 CM to stimulate PGE2 synthesis by bone marrow cells appears to result from a synergistic action between GM-CSF and IL-1.
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Florence: cytokines and T-cell subsets.
The second international congress on cytokines, held in Florence (March 23-25, 1992), has been marked by the presentation of recent data about cytokine-deficient mice obtained by homologous recombination which, contrary to expectation, exhibit normal T and B cell development. The cloning of a few new cytokines and receptors were also reported as well as recent advances in the knowledge of the oldest members of this quickly growing family. The meeting has also been marked by a great number of reports providing insights into T cell-dependent immune response. Participants provided evidence, through their different experimental models, that functional T helper dichotomy, TH1 versus TH2, was of physiological relevance both in mice and humans, while other reports highlighted the differential regulation of these T lymphocytes subsets. An interesting discussion took place about their differentiation process and the harmonization of the concepts of naive, memory and effector T cells with the T subset classification according to cytokine production profile.
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[Paracrine control of spermatogenetic stem cells: example of the leukemia inhibitory factor].
Correct regulation of spermatogonial mitosis and, more specifically, the control of the balance between differentiation and proliferation to allow renewal of the stem cell stock, are essential for the maintenance of spermatozoa production throughout life. The mechanisms underlying this control are still unknown. However, recent studies suggest that some locally produced cytokines may be involved in the regulation of spermatogonial activity. In this context, Leukemia Inhibitory Factor (LIF) exhibits interesting properties regarding stem cells and, particularly, primordial germ cells. The present study aimed at investigating LIF production and LIF binding abilities by/of the different testicular cells types (somatic and germ cells). Our study demonstrates that LIF is produced within the testis, mainly by peritubular cells which are in the vicinity of spermatogonia, the latter cells expressing high levels of LIF receptors. These results strongly suggest an involvement of LIF in the control of spermatogonial activity.
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