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.     

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.     

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.     

Stimulator of proliferation of spleen colony-forming cells in T-cell deprived mice treated with cyclophosphamide or irradiation

A role for T-cells in the regulation of CFU-S proliferation was investigated by determining the presence and activity of CFU-S proliferation stimulator (CFU-S stimulator) in adult mouse bone marrow after irradiation or cyclophosphamide (Cy) treatment. CBA mice previously deprived of T-cells by thymectomy, irradiation and bone marrow reconstitution (TIR) were thereafter treated with 4.5 Gy irradiation or 200 mg/kg Cy. Regenerating bone marrow cells of TIR and corresponding control mice after irradiation or Cy treatment produced CFU-S stimulator. The dose dependent increase in cytosine arabinoside cell death of normal bone marrow day 8 CFU-S was found when both CFU-S stimulators obtained after irradiation of TIR or corresponding control animals were tested. CFU-S stimulator activity in the bone marrow of TIR-Cy treated mice was also detected, but the effect was not dose-dependent. This was not related to the presence of an inhibitor of CFU-S proliferation. It appears that the CFU-S stimulator activity is not related to IL-6, IL-1 or IL-2, or to an inhibitor of IL-6 or IL-1 activity. The results demonstrate the existence of CFU-S proliferation stimulator unrelated to the two major monokines in the bone marrow of immunosuppressed mice.     

Cyclic AMP response to various haemopoietic regulators

Diffusible inhibitors and stimulators are involved in the regulation of bone marrow pluripotent stem cell (CFU-S) proliferation. We have previously shown the existence of CFU-S inhibitors in foetal calf marrow and liver and have started their purification. The lack of a simple and time-saving test to determine the kinetic state of CFU-S and the activity of the inhibitors led us to explore the possibility of a biochemical proliferation marker that could be used for screening purpose. Since it was shown that cyclic AMP was implicated in the regulation of CFU-S proliferation, it was of interest to study the variations in cAMP levels after stimulation and inhibition of CFU-S entry into cycle. The results of in vitro experiments showed that the increase in cAMP levels observed in bone marrow cells after incubation with different haemopoietic stimulators was specific neither for bone marrow cells nor for the various haematopoietic regulators. In the in vivo experiments, an increased cAMP level was observed 8 hr after one injection of Ara-C at the time when CFU-S are recruited into S phase. However, no modification of cAMP levels has been observed after injection of CFU-S inhibitors in the Ara-C-treated mice. Although cAMP does not seem to be a suitable marker for testing the activity of inhibitory fractions during the purification process, this work has contributed to the study of CFU-S stimulators.     

人胎肝中一种35kD成分的造血调控研究

３－５月胎龄人肝脏造血异常活跃。其中造血干细胞约有３０－４０％处于细胞周期Ｓ期,远高于成年骨髓中约１０％的比例。胎肝中存在活性很高血的造血干细胞增殖刺激因子可能是这一活跃功能的分子基础。基于这种事实,本文用小鼠ＣＦＵ－Ｓ“自杀”率对这种活性进行了检测。经过多步分离纯化,获得一分子量约３５ｋＤ的单一活性组分,定名为ＦＬＳ－４。ＦＬＳ－４作用于脐带血ＣＤ３４细胞,使其＾３Ｈ－ＴｄＲ掺入率提高近１倍,与     

Cyclic Amp Response to Various Haemopoietic Regulators

Diffusible inhibitors and stimulators are involved in the regulation of bone marrow pluripotent stem cell (CFU-S) proliferation. We have previously shown the existence of CFU-S inhibitors in foetal calf marrow and liver and have started their purification. the lack of a simple and time-saving test to determine the kinetic state of CFU-S and the activity of the inhibitors led us to explore the possibility of a biochemical proliferation marker that could be used for screening purpose. Since it was shown that cyclic AMP was implicated in the regulation of CFU-S proliferation, it was of interest to study the variations in cAMP levels after stimulation and inhibition of CFU-S entry into cycle. the results of in vitro experiments showed that the increase in cAMP levels observed in bone marrow cells after incubation with different haemopoietic stimulators was specific neither for bone marrow cells nor for the various haematopoietic regulators. In the in vivo experiments, an increased cAMP level was observed 8 hr after one injection of Ara-C at the time when CFU-S are recruited into S phase. However, no modification of cAMP levels has been observed after injection of CFU-S inhibitors in the Ara C-treated mice. Although cAMP does not seem to be a suitable marker for testing the activity of inhibitory fractions during the purification process, this work has contributed to the study of CFU-S stimulators.     

人胎肝中一种35kD成分的造血调控研究

肝脏作为胚胎发育一定阶段的造血中心器官,其造血干细胞增殖十分活跃,已发现的具有正向调节其作用的因子有IL-3,IL-6,GM-CSF,SCF及FLT 3配基等;然而上述因子的作用并不足以解释胎肝如此活跃的造血活动。推测可能还有其他特异的源于胎肝造血微环境细胞的正向调节因子存在,并以旁分泌方式作用于G_0期造血干细胞,使其进入细胞周期。     

Local regulation of haemopoietic stem cell proliferation in mice following irradiation

Changes in the kinetic state of pluripotent haemopoietic spleen colony forming cells (CFU-S) and of the CFU-S proliferation stimulator have been studied following whole-body X-irradiation. Rapid recruitment of CFU-S into cell cycle by 30 min after irradiation was observed following low doses (0.5 Gy) but a delay of 6 h occurred after higher doses (1.5 and 4.5 Gy). These changes in proliferative state correlated with the presence of the CFU-S proliferation stimulator. CFU-S irradiated in vitro in bone marrow plugs were also recruited into cycle illustrating directly the local nature of the feedback mechanism. CFU-S removed from 1.5 Gy irradiated recipients at a time when they were not in cycle were not responsive to the CFU-S proliferation stimulator. The CFU-S proliferation stimulator was produced by Ia positive cells in the irradiated bone marrow. The regulation changes occurring shortly after irradiation cannot simply be controlled by the size of the CFU-S compartment.     

Granulocyte-macrophage colony-stimulating factor is a chemoattractant cytokine for human neutrophils: involvement of the ribosomal p70 S6 kinase signaling pathway

GM-CSF stimulates proliferation of myeloid precursors in bone marrow and primes mature leukocytes for enhanced functionality. We demonstrate that GM-CSF is a powerful chemotactic and chemokinetic agent for human neutrophils. GM-CSF-induced chemotaxis is time dependent and is specifically neutralized with Abs directed to either the ligand itself or its receptor. Maximal chemotactic response was achieved at approximately 7 nM GM-CSF, and the EC(50) was approximately 0.9 nM. Both concentrations are similar to the effective concentrations of IL-8 and less than the effective concentrations of other neutrophil chemoattractants such as neutrophil-activating peptide-78, granulocyte chemotactic protein-2, leukotriene B(4), and FMLP. GM-CSF also acts as a chemoattractant for native cells bearing the GM-CSF receptor, such as monocytes, as well as for GM-CSF receptor-bearing myeloid cell lines, HL60 (promyelomonocyte leukemic cell line) and MPD (myeloproliferative disorder cell line), following differentiation induction. GM-CSF induced a rapid, transient increase in F-actin polymerization and the formation of focal contact rings in neutrophils, which are prerequisites for cell migration. The mechanism of GM-CSF-induced chemotaxis appears to involve the cell signaling molecule, ribosomal p70 S6 kinase (p70S6K). Both p70S6K enzymatic activity and T(421)/S(424) and T(389) phosphorylation are markedly increased with GM-CSF. In addition, the p70S6K inhibitor hamartin transduced into cells as active protein, interfered with GM-CSF-dependent migration, and attenuated p70S6K phosphorylation. These data indicate that GM-CSF exhibits chemotactic functionality and suggest new avenues for the investigation of the molecular basis of chemotaxis as it relates to inflammation and tissue injury.     

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.     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

Proliferation and splenic migration of mouse hematopoietic stem cells following a single injection of Mycoplasma arthritidis

The effect of a single injection of live M. arthritidis microorganisms on the bone marrow and spleen CFU-S populations was assessed. One of the principal findings is that marrow CFU-S are recruited into cell cycle (as determined by hydroxyurea kill) early after M. arthritidis administration and stay in the cycle for at least 2 weeks thereafter. The peak level of cycling value (47%) was observed on day 4. The duration of increased CFU-S cycling activity was shown to coincide with a time period during which a significant rise in the number of endogenous CFU-S was maintained. The other important finding is that splenic seeding efficiency ("f-factor") declines by 56% one day following M. arthritidis administration. The latter effect could be attributed to the binding of mycoplasmas to the surface of CFU-S as specific rabbit antiserum against M. arthritidis incubated in vitro with the bone marrow cells of infected donor mice caused an up to 48% reduction in the in vivo colony-forming ability of CFU-S.     

Anti-tumor cytotoxic potential and effect on human bone marrow GM-CFU of human LAK cells generated in response to various cytokines.

The effects of various recombinant cytokines i.e. IL-1 alpha, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha and GM-CSF used either alone or in combination with IL-2, were investigated in this study. First, their capacity to induce killer cells from human PBL was examined by evaluating the degree of killing of human NK-sensitive K562 or NK-resistant Daudi cells. Second the effects of these cytokines, LAK cells (at 1/1, 2/1, 4/1 ratio LAK effectors/bone marrow cell targets) and of the supernatants from washed killer cell cultures, were examined on the colony forming ability of human bone marrow for GM-CFU in vitro. Various degrees of NK activity against K562 was observed in PBL stimulated with the cytokines, whereas LAK activity was found only with IL-2 alone. Culture of PBL with IL-2 + IL-1 alpha or IL-2 + IL-6 or IL-2 + GM-CSF resulted in the highest LAK killing. However, addition of TNF-alpha, or IFN-gamma to IL-2 in cultures resulted in a significant suppression of LAK cell activity. Addition of IL-1 alpha, IL-2, IL-3, and IL-4 to BM cultures had little or no effect on day 14 GM-CFU, whereas addition of IL-6 and GM-CSF resulted in a stimulatory effect. LAK cells induced with IL-2 alone had no significant suppressive effects on GM-CFU.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and characterization of a B cell stimulatory factor (BSF-TC) derived from a bone marrow stromal cell line

Stromal cell lines derived from murine bone marrow support the growth of immature pre-B cells and produce cytokines that affect the growth and differentiation of other hematopoietic precursors. Conditioned medium (CM) from one such line (TC-1) stimulated marked proliferation of B cells previously activated by anti-Ig (anti-Ig blasts). Proliferation of anti-Ig blasts was not induced by purified cytokines known to be produced by TC-1 (CSF-1, GM-CSF, or G-CSF) or by IL-1, IL-2, IL-3, IL-4, IL-5, or IL-6. Furthermore, IL-2, IL-4, and IL-5, alone or in combination, failed to support proliferation or differentiation of anti-Ig blasts. TC-1 CM enhanced proliferation of B cells that were co-cultured with LPS, anti-Ig, or dextran sulfate; co-stimulation with anti-Ig was unaffected by the presence of monoclonal anti-IL-4. Proliferation of low, but not high, density B cells isolated from spleen was directly stimulated by TC-1 CM. These results suggest that bone marrow stromal cells produce a novel B cell stimulatory factor (BSF-TC) that induces proliferation of activated B cells.     

Genetic factors influencing murine hematopoietic productivity in culture

In order to study a previously described genetic difference manifested in stem cell kinetics of specific mouse strains, effects of this putative gene, stk, were measured on growth and expansion of stem and progenitor cell populations ex vivo. Bone marrow cells from each of two inbred mouse strains, C57BL/6J and DBA/2J, were placed into separate bioreactor cultures perfused continuously with growth medium containing erythropoietin (Epo), interleukin-3 (IL-3), granulocyte-macrphage colony stimulating factor (GM-CSF), and Kit ligand as well as 5% CO₂. Expansion of cell numbers reached 20-fold for DBA/2J and 10-fold for C57BL/6J marrow within about 1 week of culture. Significant production was also seen of colonyforming unit (CFU)-GM (up nine-fold from input levels) just prior to the cell production peak, and, importantly, moderate expansion of day 12 colony-forming unit-spleen (CFU-S; two- to threefold) occurred as well, although CFU-S production peaked at a relatively short 4 days. CFU-S and CFU-GM levels declined rapidly in culture, either because of unfavorable growth conditions or terminal differentiation. Attempts to remove toxic metabolites by increasing the media perfusion rate resulted in a boost in cell expansion capability by DBA/2J marrow. In bioreactors in which stromal cells were established before marrow inoculation, there was greater expansion of CFU-S (especially by DBA/2J) and CFU-GM, although total cell yield appeared to be unaffected, perhaps because the maximum cell density had already been reached. The relative high potential for CFU-S expansion measured in DBA/2J marrow over that of C57BL/6J will be useful in following genetic contributions to bone marrow production capacity. © 1995 Wiley-Liss, Inc.     

Stimulator of proliferation of spleen colony-forming cells in acute sterile inflammation

The presence and activity of a spleen colony-forming cell (CFU-S) proliferation stimulator was investigated in rat bone marrow after induction of sterile inflammation. Wistar rats were treated intraperitoneally with two 15 ml injections of 3.5% polyvinylpyrrolidone (PVP) at 18 h intervals, and the presence of CFU-S proliferation stimulator determined in bone marrow 6, 20 and 24 h after the first injection. The marrow of these mice was used to condition medium which was then fractionated using Amicon Diaflo ultrafiltration membranes. The 30–50 kDa fraction, taken from 20 h post-PVP-bone marrow extract, was found to induce cycling of d8-CFU-S in normal mouse bone marrow. This activity was not related to the presence of interleukins-1, -2 or -6 like activities in the material tested. The results demonstrate the existence of CFU-S proliferation stimulator in the bone marrow of rats with sterile inflammation (i.e. in an in vivo tissue response to non-specific cell stimulation), similar to that originally described as a macrophage product in mouse bone marrow after treatment with a variety of cytotoxic agents.     

Haemopoietic stem cell proliferation in the bone marrow of S1/S1d mice

In marrow from Sl/Sld mice (but not +/+ mice) day 7 and day 8 CFU-S proliferate whilst day 10 and day 12 CFU-S exhibit negligible proliferation. Media conditioned by both +/+ and Sl/Sld marrow contains an inhibitor of CFU-S proliferation but day 8 CFU-S in +/+ and Sl/Sld marrow show marked dose-response differences to this factor. To inhibit the proliferation of Sl/Sld CFU-S required approximately ten times the concentration of inhibitor that inhibited the proliferation of +/+ CFU-S. Thus abnormally responsive day 8-CFU-S were shown to proliferate in an inhibitory environment. Abnormalities in Sl/Sld CFU-S function were also demonstrated in heterotopic transplantation experiments using +/+ and Sl/Sld donors and hosts to obtain ectopic bone marrow with various stromal (donor) and haemopoietic (host) combinations. Day 8 Sl/Sld CFU-S were seen to proliferate, irrespective of whether the stromal environment was derived from Sl/Sld or +/+ marrow. Sl/Sld mice are generally regarded as animals in which there is a genetically determined defect in haemopoiesis due to an abnormality in the haemopoietic environment. It is difficult, however, to attribute the abnormal CFU-S behaviour in these experiments to environmental factors and the results are consistent with mutation at the Sl locus affecting the responses of CFU-S to regulatory signals, i.e. the genetic defect is not confined to the stromal environment.     

Cells with marrow and spleen repopulating ability and forming spleen colonies on day 16, 12, and 8 are sequentially ordered on the basis of increasing rhodamine 123 retention

Mouse bone marrow cells (BMC) were subjected to countercurrent centrifugal elutriation and subsequently separated on the basis of light scatter and fluorescence intensity after being labeled with the supravital dye Rhodamine 123 (Rh-123). The sorted cells were then assayed for their in vivo spleen colony-forming ability (day -8, -12, and -16 CFU-S) and their ability to repopulate the bone marrow or spleen over a 13-day period with CFU-S-12, CFU-GM, or nucleated cells. Cells with marrow repopulating ability (MRA), as measured by the ability of the sorted cells to repopulate the marrow with secondary CFU-S-12 or CFU-GM, had low affinity for Rh-123. These cells showed minimal spleen colony-forming ability, and the ratio of MRA to CFU-S-12 in this preparation was 309. Cells with spleen repopulating ability (SRA), CFU-S-16, CFU-S-12, and CFU-S-8 retained increasing amounts of Rh-123, respectively, and CFU-S-8 were almost exclusively found among cells with high Rh-123 affinity. These cells also included about half of all day-12 CFU-S, and the ratio of MRA to day-12 CFU-S was 0. The results show that MRA cells, SRA cells, CFU-S-16, CFU-S-12, and CFU-S-8 can be sequentially ordered on the basis of increasing mitochondrial activity. The data also demonstrate for the first time, and without the application of negative selection by the use of cytostatic agents, that MRA cells are a separate class of primitive hemopoietic stem cells that fully meet the criteria of pre-CFU-S.