Eµ and 3'RR IgH enhancers show hierarchic unilateral dependence in mature B-cells.

Enhancer and super-enhancers are master regulators of cell fate. While they act at long-distances on adjacent genes, it is unclear whether they also act on one another. The immunoglobulin heavy chain (IgH) locus is unique in carrying two super-enhancers at both ends of the constant gene cluster: the 5'Eµ super-enhancer promotes VDJ recombination during the earliest steps of B-cell ontogeny while the 3' regulatory region (3'RR) is essential for late differentiation. Since they carry functional synergies in mature B-cells and physically interact during IgH locus DNA looping, we investigated if they were independent engines of locus remodelling or if their function was more intimately intermingled, their optimal activation then requiring physical contact with each other. Analysis of chromatin marks, enhancer RNA transcription and accessibility in Eµ- and 3'RR-deficient mice show, in mature activated B-cells, an unilateral dependence of this pair of enhancers: while the 3'RR acts in autonomy, Eµ in contrast likely falls under control of the 3'RR.

PAF and haematopoiesis: III. Presence and metabolism of platelet-activating factor in human bone marrow.

Platelet-activating factor (PAF) is a phospholipid compound with major immunoregulatory activities. The present study shows that human bone marrow contains 576 +/- 39 pg PAF/ml (n = 35). Bone marrow-derived PAF exhibits the same biophysical and biological properties that synthetic PAF. PAF concentrations in bone marrow are correlated with the granulocyte (r = 0.4, P = 0.02) but not with the lymphocyte (r = 0.24, P = 0.17) and the monocyte (r = 0.12, P = 0.48) counts. In bone marrow PAF is inactivated by a plasma PAF acetylhydrolase activity (48.0 +/- 2.3 nmol/min per ml, n = 34). Experiments with [3H]PAF indicate that human bone marrow cells actively metabolize this potent molecule by the deacetylation-transacylation pathway. Results of this investigation indicate the permanent presence of significant amounts of PAF in bone marrow suggesting its putative involvement in the processes of bone marrow cell proliferation and maturation.

Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.

Arachidonic acid and human bone marrow stromal cells.

Human bone marrow stromal cells regulate the growth of marrow hematopoietic progenitors by secreting cytokines. Arachidonic acid (AA) is the fatty acid precursor of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) that modulate the growth of human bone marrow progenitors. We have investigated the incorporation of AA in human bone marrow stromal cell cultures, their production of PGE2 and LTB4 and the effect of AA on their growth. Gas chromatography analysis reveals the presence of AA in the human bone marrow plasma and in bone marrow stromal cell cultures. In stromal cells, [3H]-AA is incorporated into triglycerides and is later delivered into phospholipids. Prelabeling-chase experiments indicate a preferential incorporation of AA into phosphatidylethanolamine and no trafficking of labeled AA between phospholipid species. Bone marrow stromal cells release PGE2 and LTB4 in response to phorbol myristic acetate (PMA) (1 microM) and tumor necrosis factor alpha (TNF-alpha) (10 ng/ml). Exogenous AA (up to 1 microM) has no significant effect on cell growth. In conclusion, human bone marrow stromal cells capt exogenous AA and, thus, may participate to the control of marrow AA concentrations. They may also regulate human marrow hematopoiesis by secreting AA metabolites such as PGE2 and LTB4.

PAF and haematopoiesis. X. Macrophage colony-stimulating factor and granulocyte macrophage colony-stimulating factor enhance platelet-activating factor acetylhydrolase production by human blood-derived macrophages.

Platelet-activating factor (PAF), a phospholipid autacoid with potent regulatory functions, is synthesized by stimulated monocytes. Macrophages are a source of the plasma acetylhydrolase activity (AHA) which regulates PAF concentrations. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are involved in the differentiation and functions of cells from the monocytic/macrophagic lineage. This work reports that M-CSF and GM-CSF stimulated AHA production by human blood monocyte-derived macrophages in a time- and dose-dependent manner. After 7 days of culture without serum, a 6- and 4-fold increase was found in cells treated with M-CSF (1000 U/ml) and GM-CSF (50 ng/ml), respectively. M-CSF (up to 1000 U/ml) and GM-CSF (up to 10 ng/ml) did not induce PAF production by human blood monocytes. While GM-CSF (10 ng/ml) and interleukin-1 (10 U/ml) stimulated M-CSF production from monocyte-derived macrophages, PAF did not. These results indicate that M-CSF and GM-CSF enhance AHA production by human blood-derived macrophages cultured in low serum concentrations. Clearly the effects of growth factors on AHA production in vivo deserve to be assessed.

Interleukin-4 (IL-4), but not IL-10, regulates the synthesis of IL-6, IL-8 and leukemia inhibitory factor by human bone marrow stromal cells.

Leukemia inhibitory factor (LIF), interleukin 6 (IL-6) and IL-8 are important regulators of inflammation and hematopoiesis. Human bone marrow stromal cells regulate marrow hematopoiesis by secreting cytokines. By using reverse-transcriptase polymerase chain reaction (RT-PCR), we demonstrate that human bone marrow stromal cells constitutively express LIF, IL-6 and IL-8 transcripts. By using specific ELISAs, we found that their spontaneous productions of LIF, IL-6 and IL-8 are elevated in response to serum and after stimulation with the pro-inflammatory cytokines IL-1alpha and TNF-alpha. The anti-inflammatory cytokine IL-4 reduces their serum- and cytokine-induced LIF secretion. By contrast, IL-4 stimulates their serum- and IL-1alpha-induced IL-6 synthesis. IL-4 has no effect on the serum-induced IL-8 synthesis by marrow stromal cells, but stimulates their cytokine-induced IL-8 production. The anti-inflammatory cytokine IL-10 has no effect on the serum- and cytokine-induced LIF, IL-6 and IL-8 synthesis by bone marrow stromal cells. RT-PCR experiments reveal the presence of IL-4 receptor alpha-chain mRNA and IL-10 receptor mRNA in cultured bone marrow stromal cells. The differential regulation by IL-4 of two related cytokines, such as LIF and IL-6, and the enhanced effect of this 'anti-inflammatory' cytokine on IL-6 and IL-8 synthesis highlight the tightly controlled regulation and the complexity of the cytokine production within the human bone marrow.

Production, metabolism and effect of platelet-activating factor on the growth of the human K562 erythroid cell line.

The human immature K562 erythroid cell line was studied for its capacity to produce and to metabolize the phospholipid molecule platelet-activating factor (PAF). K562 cells produced PAF under calcium ionophore stimulation. Lyso PAF and acetyl-CoA (the acetate donor molecule for the acetylation of lyso PAF into PAF) had no effect on the amounts of PAF produced by ionophore-stimulated cells. The metabolism of PAF and lyso PAF by K562 cells was compared to that of freshly-isolated human bone marrow erythroblasts and blood erythrocytes. K562 cells rapidly metabolized [3H]PAF and [3H]lyso PAF with 1-alkyl analogue of phosphatidylcholine as the major metabolic product. In contrast, blood erythrocytes did not. PAF acetylhydrolase activity levels in K562 cells and bone marrow erythroblasts were similar and higher than in blood erythrocytes. PAF (1-100 nM) stimulated [3H]thymidine incorporation in K562 cells grown in low serum concentration, a non-metabolizable PAF agonist being more potent than PAF to stimulate thymidine incorporation. PAF receptor mRNA was detected in K562 cells by polymerase chain reaction on reverse transcripts. The present study demonstrates that K562 cells produce and metabolize PAF and underlines the putative role of erythroid precursors in the modulation of bone marrow PAF concentrations. The effect of PAF on the growth of K562 cells might be mediated through PAF receptors suggesting a potential role of PAF on the proliferation and functions of human erythroid marrow precursors.

Cooperation between platelets and neutrophils for paf-acether (platelet-activating factor) formation.

Association of platelets and neutrophils is frequently observed within thrombi or inflammatory sites. Interactions between these two cell populations have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether, which is synthesized by activated platelets and neutrophils. Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelet and neutrophil cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 x 10(6)/ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 +/- 2.5 ng/ml paf-acether. Human washed platelets (3 x 10(8)/ml) stimulated with thrombin (1 IU/ml) formed 0.60 +/- 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as did platelets and neutrophils separately (10.90 +/- 4.25 ng/ml, n = 6, P less than .001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled (10.24 +/- 3.81 ng/ml paf-acether vs. 5.30 +/- 2.23, P less than .05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to an increase of biosynthesis of paf-acether by neutrophils (13.86 +/- 2.26 ng/ml paf-acether vs. 5.76 +/- 0.38, P less than .05, n = 3). These results indicate that a cooperation between platelets and neutrophils exists for paf-acether formation. The phenomenon depends on a platelet-derived soluble factor, possibly lyso paf-acether. This cell-to-cell interaction is of interest since paf-acether is formed by and acting on platelets and neutrophils and represents a molecular basis for potent amplification of inflammatory reactions.

PAF and hematopoiesis. VIII. Biosynthesis and metabolism of PAF by human bone marrow stromal cells.

Human bone marrow stromal cells were studied for their ability to synthesize and to metabolize platelet-activating factor (PAF), a lipidic compound with potent immunoregulatory properties. When stimulated with 2 microM calcium ionophore for 60 minutes, cultures of stromal cells increased their PAF production (3.52 +/- 0.91 ng/1 x 10(6) cells) compared with controls (0.82 +/- 0.13 ng/1 x 10(6) cells). Addition of exogenous lyso PAF (100 nM) and acetyl-CoA (100 microM) during calcium ionophore stimulation did not change the PAF production. The synthesis of PAF was not influenced by the concentration of albumin in the incubation buffer. The PAF from stromal cells exhibited a hexadecyl chain at the sn-1 position of the molecule, as determined by reverse-phase HPLC. While stromal cells contained low amounts of PAF acetylhydrolase activity and did not secrete it in the culture medium, they metabolized exogenous PAF with 1-alkyl-2-acyl-glycero-phosphocholine and neutral lipids as the major metabolic products. The present results are the first to demonstrate the synthesis and metabolism of PAF by human bone marrow stromal cells. These data suggest that they might be a source of the PAF found in the human bone marrow and/or might be important in the regulation of its levels. The role of PAF on the proliferation and functions of human hematopoietic cells deserves investigation.

Production of paf-acether by human epidermal cells.

The production of the inflammatory mediator paf-acether (paf) from human epidermal cells was investigated in vitro. Human epidermal cells, freshly isolated from normal skin or in culture, were incubated in Tyrode's buffer containing 0.25% lipid-free bovine serum albumin in the presence of 2 microM calcium ionophore A23187, at 37 degrees C, for 1 to 60 min. Paf production slightly began at the first min of stimulation, was significant after 10 min, reached a maximum at 20 min (251 +/- 25 pg/l X 10(6) cells, mean +/- 1 SD), and decreased thereafter. About 50% of the paf amount produced by epidermal cells was recovered in supernatants. Addition of the non-acetylated paf precursor 1-O-octadecyl-sn-glycero-3-phosphocholine, i.e., lyso-paf, at 0.1 microM to epidermal cells during A23187-stimulation did not alter this production. In contrast, addition of acetyl-coenzyme A at 0.1 mM enhanced paf production by 5 times. The material produced by epidermal cells was identical to synthetic paf because: 1) the aggregation of aspirin-treated and ADP-insensitive washed rabbit platelets it induced was inhibited by BN 52021, an antagonist of the paf putative receptor; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse-phase (RP) high-pressure liquid chromatography (HPLC) elution. The paf precursors, i.e., lyso-paf and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, were also detected in epidermal cells, stimulated with A23187 or not. As determined by RP-HPLC analysis and confirmed by gas chromatography analysis, these precursors and the paf produced by epidermal cells exhibited more than 90% of a hexadecyl chain at the sn-1 position of the molecule. The present results demonstrate the synthesis and release of paf by normal human epidermal cells. Paf production within the epidermis might account for the development of cutaneous inflammation and the pathogenesis of many skin disorders.

Presence of paf-acether in human thymus.

Paf-acether (paf) is a phospholipid mediator of inflammation endowed with major immunoregulatory properties. The present study demonstrates that human thymus contains large amounts of paf, as well as paf precursors. In addition, isolated thymic cells produced paf under ionophore stimulation. Paf from thymus exhibited the same biological and physiochemical properties as synthetic paf. The purity and molecular structure of paf from thymus were further characterized by reverse-phase HPLC and gas chromatography with electron-capture detection. These findings may have important implications since thymus microenvironment is essential in the proper development of bone marrow progenitors committed to the T cell lineage into thymocytes capable of emigrating to the periphery as functional T lymphocytes.

Synthesis of paf-acether from exogenous precursors by the prokaryote Escherichia coli.

Paf-acether (paf) is a potent mediator of inflammatory diseases and septic shock. Using normal-phase HPLC, a paf-like activity was found in culture supernatants from E. coli. Prokaryotic paf exhibited the same biological and physico-chemical properties as eukaryotic cells and synthetic paf. Further, reverse-phase HPLC indicates that paf generated by bacteria is predominantly of the hexadecyl and octadecyl species. When cultures were supplemented with lyso-paf, a dramatic increase in paf production was observed. The purity and molecular structure of bacterial paf were further characterized by mass spectral analysis. These results could be of importance considering the pathogenetic role of enterobacteria. Further, it appears that the competence to form and release paf is an early phylogenetic development.

Incorporation of arachidonic acid in a human cancer gastric tumor cell line (HGT) at various stages of cell proliferation.

Human cancer gastric cells (HGT) in culture were labeled with [14C]arachidonic acid. The kinetic of acylation of arachidonic acid was similar using cells after 3, 5 and 7 days of culture. However, the duration of the proliferation enhanced the acyltransfer to triglycerides and inhibited the transfer to phospholipids. After 8 h of incubation with [14C]arachidonic acid the radioactivity was predominantly recovered in the phosphatidylcholine and phosphatidylethanolamine species. Prelabeling-chase experiments indicated a preferential incorporation of arachidonic acid into phosphatidylcholine followed by a transfer of arachidonate to phosphatidylethanolamine.

Effects of eicosanoid metabolism inhibitors on growth of a human gastric tumour cell line (HGT).

HGT cells are a human gastric cell line derived from a tumour of the stomach. We have investigated the effects of lipoxygenase and cyclooxygenase metabolism inhibitors on HGT cell proliferation, on fatty acid composition of HGT cells and on the incorporation and distribution of arachidonic acid (AA) in HGT lipids. The cyclooxygenase inhibitor aspirin and both cyclooxygenase and the lipoxygenase inhibitor BW 755C suppressed cell proliferation in a concentration-dependent manner. The inhibition of HGT proliferation did not result from a modulation of the fatty acid composition of membrane lipid, which was not affected by treatment with the various inhibitors. Inhibitors of AA metabolism did not alter acylation of exogenous AA into HGT cells nor its subsequent distribution in the lipid and phospholipid species. The role of cyclooxygenase eicosanoids in HGT proliferation is discussed.

The decreased transfer of arachidonic acid from triglycerides to phospholipids during proliferation of a human gastric tumor (HGT) cell line is not linked to modification of membrane fluidity.

Prelabeling-chase experiments with [14C]arachidonic acid in human cancer gastric cells (HGT) indicated a preferential incorporation of arachidonic acid into triglycerides followed by a transfer to phospholipids. This phenomenon was quicker using cells in log phase growth than in quiescent phase and was not linked to modification of HGT membrane fluidity assessed by the fluorescence polarization of the 1,6-diphenyl hexatriene probe. By contrast, growing cells in a medium enriched with oleic or linoleic acids increase plasma membrane fluidity. The importance of the site of arachidonic incorporation in HGT lipid is discussed.

PAF and hematopoiesis. II. Elevated levels of plasma paf acetylhydrolase after rapid infusion of 5-fluorouracil in cancer patients.

Blood platelet-activating factor (PAF) levels are regulated by a plasma PAF acetylhydrolase. We investigated its levels in cancer patients during the course of a 5-day 5-fluorouracil (5-FU) treatment. PAF acetylhydrolase increased in nine patients with daily bolus infusion of 0.4 g 5-FU per m2 of body surface (81.7 +/- 8.7 nmol PAF/min/ml vs. 66.6 +/- 7.0; P < 0.001 for day 5 as compared to day 1). By contrast PAF acetylhydrolase did not change in seven patients with continuous infusion of 5-FU. The meaning of these results is discussed in respect of the immunoregulatory role of PAF.

PAF and haematopoiesis XII: macrophage colony-stimulating factor (M-CSF) decreases platelet-activating factor (PAF) acetylhydrolase production in macrophagic J774 cells.

Platelet-activating factor (PAF) is a phospholipid mediator with major immunoregulatory activities. Macrophages produce PAF acetylhydrolase activity, which regulates blood PAF concentrations. Macrophage colony-stimulating factor (M-CSF) is involved in the differentiation and functions of cells from the monocytic/macrophagic lineage. We found that murine macrophagic J774 cells metabolized PAF with lyso PAF as the major metabolite product. As in mouse plasma, the metabolism of PAF by J774 cells was not inhibited by PMSF, p-BPB, DTNB and quinacrine, M-CSF (100-5000 U/ml) significantly decreased PAF acetylhydrolase activity of the J774 cell without exhibiting a significant effect on cell growth. Elevated concentrations of M-CSF are found in blood and tissues during inflammatory states. It could be suggested that a decreased PAF catabolism by tissue macrophages in response to M-CSF may induce local elevated PAF concentrations, thus amplifying the inflammatory response.

Incorporation and effect of arachidonic acid on the growth of the human K562 cell line.

Lipoxygenase inhibitors reduce the growth of K562 cells (chronic myelogenous human leukaemia blasts) suggesting a role for endogenous lipoxygenase products of arachidonic acid (AA) in their proliferation. The objectives of this work are to investigate the incorporation of AA into K562 cells and to assess the effects of the exogenous addition of AA and lipoxygenase products on their growth. The mechanism of acylation of [3H]-AA indicates that K562 cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]-AA is incorporated unmodified into K562 phospholipids and is linked by an ester bond. Prelabelling-chase experiments indicate a transfer of labelled AA from phosphatidylcholine to phosphatidylethanolamine. The addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin B4, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE) has no effect on K562 cell proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while K562 cells readily incorporate AA into their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

PAF and haematopoiesis. IV. Modifications of spleen and thymus PAF contents after a single dose of the chemotherapeutic drug 5-fluorouracil in mice.

The spleen and thymus of mice were examined for the presence of PAF after injection of 5-fluorouracil (5-FU) (200 mg/kg). A significant increase of the spleen (P = 0.005) and thymus (P < 0.05) PAF concentrations was noted 48 h after 5-FU infusion. PAF levels in thymus are similar to those of controls from days 4 to 14. By contrast, spleen PAF significantly decreased (0.005 < P < 0.03) from days 7 to 14. Conversely, the 5-FU administration did not modify the spleen and plasma acetylhydrolase activity, suggesting that the variations of PAF levels in thymus and spleen were mainly due to differences of local PAF production. Thus, the chemotherapeutic drug 5-FU modulates in vivo PAF production in haematopoietic organs of mice. Considering the effects of PAF in the processes of B- and T-cell proliferation and functions, these results could be of importance for the role of PAF during human cancer therapy and haematopoiesis in vivo.