Interleukin 13: novel role in direct regulation of proliferation and differentiation of primitive hematopoietic progenitor cells.

The recently cloned interleukin 13 (IL-13) shares most investigated biological activities on B lymphocytes and monocytes with IL-4. In this study we investigated for the first time the potential role of IL-13 in the regulation of the growth of hematopoietic progenitor cells. IL-13 enhanced stem cell factor (SCF)-induced proliferation of Lin-Sca-1+ bone marrow progenitor cells more potently than IL-4. The effect of IL-13 was purely synergistic, since IL-13 alone stimulated no colony formation. Single cell experiments suggested that the synergistic effect of IL-13 on Lin-Sca-1+ progenitors was directly mediated. In contrast, IL-13 had no synergistic activity on SCF-induced proliferation of the more mature Lin-Sca-1- progenitor cells. Thus, the cloning frequency in response to SCF + IL-13 was at least 20-fold higher in the Lin-Sca-1+ than the Lin-Sca-1- progenitor cell population. Furthermore, IL-13 but not IL-4 synergistically enhanced colony formation of Lin-Sca-1+ progenitors in response to granulocyte/macrophage colony-stimulating factor (GM-CSF) (threefold), whereas both IL-4 and IL-13 enhanced G-CSF-induced colony formation (threefold), and neither of the two significantly affected CSF-1 and IL-3-induced proliferation. Finally, whereas stimulation of Lin-Sca-1+ progenitors by SCF + G-CSF resulted in the formation of 90% granulocytes, the addition of IL-13 resulted in the production of macrophages exclusively. This novel effect on differentiation was directly mediated, shared with IL-4, and could not be observed on Lin-Sca-1- progenitor cells. Collectively, these findings indicate a novel role of IL-13 in early myelopoiesis, partially overlapping but also different from that of IL-4.

The FLT3 ligand potently and directly stimulates the growth and expansion of primitive murine bone marrow progenitor cells in vitro: synergistic interactions with interleukin (IL) 11, IL-12, and other hematopoietic growth factors.

The recently cloned murine flt3 ligand (FL) was studied for its ability to stimulate the growth of primitive (Lin-Sca-1+) and more committed (Lin-Sca-1-) murine bone marrow progenitor cells, alone and in combination with other hematopoietic growth factors (HGFs). Whereas FL was a weak proliferative stimulator alone, it potently synergized with a number of other HGFs, including all four colony-stimulating factor (CSF), interleukin (IL) 6, IL-11, IL-12, and stem cell factor (SCF), to promote the colony formation of Lin-Sca-1+, but not Lin-Sca-1- or erythroid progenitor cells. The synergistic activity of FL was concentration dependent, with maximum stimulation occurring at 250 ng/ml, and was observed when cells were plated at a concentration of one cell per culture, suggesting that its effects are directly mediated. 2 wk of treatment with FL in combination with IL-3 or SCF resulted in the production of a high proportion of mature myeloid cells (granulocytes and macrophages), whereas the combination of FL with G-CSF, IL-11, or IL-12 resulted predominantly in the formation of cells with an immature blast cell appearance. Accordingly, FL in combination with G-CSF or IL-11 expanded the number of progenitors more than 40-fold after 2 wk incubation. Thus, FL emerges as a potent synergistic HGF, that in combination with numerous other HGFs, can directly stimulate the proliferation, myeloid differentiation, and expansion of primitive hematopoietic progenitor cells.

All-trans- and 9-cis-retinoic acid: potent direct inhibitors of primitive murine hematopoietic progenitors in vitro.

Retinoic acid (RA) stimulates the clonal proliferation of mature bone marrow progenitor cells and inhibits the growth of leukemic progenitors, whereas its effects on normal primitive hematopoietic progenitors have not yet been investigated. This study investigated the ability of all-trans- and 9-cis-RA to modulate the proliferation and differentiation of murine Lin-Sca-1+ bone marrow progenitor cells. Both RA isoforms inhibited in a reversible and dose-dependent fashion, the proliferation of multi- but not single-factor responsive Lin-Sca-1+ progenitor cells. The 50% effective dose was 10 nM for both all-trans- and 9-cis-RA. Maximum inhibition was observed at 100-1,000 nM RA, resulting in a 50-75% reduction in the number of proliferative clones. Lin-Sca-1+ cells with high proliferative potential were preferentially inhibited by RA, resulting in a 80-100% inhibition depending on the hematopoietic growth factors stimulating their growth. The inhibitory effects of RA were directly mediated on the target cell, since the effects were observed at the single cell level. Furthermore, autocrine transforming growth factor beta (TGF-beta) production can probably not account for the observed inhibitory effects of RA, since a TGF-beta neutralizing antibody did not block RA-induced inhibition. Whereas RA, in general, is a differentiation-inducing agent, treatment of Lin-Sca-1+ progenitors resulted in the accumulation of an increased fraction of blasts and immature myeloid cells. Thus, RA inhibits the proliferation as well as differentiation of normal primitive hematopoietic progenitor cells.

Distinct and direct synergistic effects of IL-1 and IL-6 on proliferation and differentiation of primitive murine hematopoietic progenitor cells in vitro.

While interleukin-1 (IL-1) and IL-6 have been demonstrated to synergize with colony-stimulating factors (CSFs) and stem cell factor (SCF) to stimulate myeloid colony formation of primitive hematopoietic progenitor cells, it has not yet been established whether these effects are directly mediated. In the present study, direct effects of IL-1 and IL-6 were examined on primitive Lin-Sca-1+ murine bone-marrow progenitor cells that were cultured and plated individually. IL-1 and IL-6 showed not only overlapping, but also distinct, patterns of direct synergy. While IL-1 or IL-6 had no proliferative effects as single growth factors, IL-1, in combination with granulocyte-macrophage CSF (GM-CSF), IL-3, CSF-1, and SCF, but not granulocyte-CSF (G-CSF), enhanced the cloning frequency of Lin-Sca-1+ progenitors three- to five-fold, whereas IL-6 increased the cloning frequency in response to all four CSFs and SCF two- to seven-fold. In all cases, the size of the colonies observed were increased as well. Furthermore, the combined action of IL-1 and IL-6 resulted in additive or synergistic enhancement of CSF- and SCF-stimulated colony formation of Lin-Sca-1+ high proliferative potential colony-forming cells (HPP-CFCs). Finally, IL-6, but not IL-1, enhanced the number of immature blast cells observed in CSF- and SCF-stimulated cultures.

Acetylsalicylic acid and sodium salicylate inhibit LPS-induced NF-kappa B/c-Rel nuclear translocation, and synthesis of tissue factor (TF) and tumor necrosis factor alfa (TNF-alpha) in human monocytes.

We have investigated the effects of acetylsalicylic acid and sodium salicylate on the LPS-induced synthesis of the pro-coagulant protein tissue factor (TF) and the pro-inflammatory protein tumor necrosis factor-alpha (TNF-alpha), as well as the prostaglandin PGE2 in human monocytes. Both drugs dose-dependently inhibited LPS-induced TF and TNF-alpha synthesis at the mRNA and the protein level, and reduced PGE2 production. As evidenced by electro mobility shift assay (EMSA) and the use of a NF-kappa B prototypic probe, these drugs probably exert their inhibitory effects by interference with the nuclear translocation of NF-kappa B/c-Rel proteins. These data may expand the understanding of the anti-thrombotic and anti-inflammatory effects of these drugs when activation of monocytes occurs.

Inhibition of IL-1 induced tissue factor (TF) synthesis and procoagulant activity (PCA) in purified human monocytes by IL-4, IL-10 and IL-13.

Exposure of monocytes to pro-inflammatory cytokines or lipopolysaccharide (LPS) may induce synthesis and expression of tissue factor (TF). In this paper we have focused on the induction of TF-activity in human monocytes by the pro-inflammatory cytokines recombinant human interleukin 1 (rhIL-1 alpha) (rhIL-1 beta) (rhIL-6) and human tumour necrosis factor alpha (rhTNF-alpha), measured as procoagulant activity (PCA) in a microtitre plate-based clot assay. In addition we have studied the modulation of IL-1 alpha/beta induced TF-mRNA and PCA by rhIL-4, rhIL-10 and rhIL13. IL-1 alpha and IL-1 beta induced a concentration dependent increase in TF-activity. Neither IL-6 nor TNF-alpha gave rise to procoagulant activity at the concentrations tested (0.2-20 ng/ml). IL-4, IL-10 and IL-13, all effectively diminished IL-1 alpha/beta induced PCA, shown at the protein- and at the mRNA-level, while cell viability was unaffected. These results add to the previously demonstrated role of IL-4 and IL-10 as inhibitors of LPS-induced TF-activity, showing that these anti-inflammatory cytokines are not specific for LPS-activation but interfere with other stimulating substances such as IL-1, which may be involved in diseases where LPS is not present.

Heparin coating reduces cell activation and mediator release in an in vitro venovenous bypass model for liver transplantation.

We used an in vitro model for venovenous bypass in a prospective, randomized study to analyze the effect on leukocytes cell activation after coating the total blood contact surface with covalently bound heparin. In ten experiments heparin-coated circuits were used, and in ten other experiments noncoated circuits were used. Monocyte cytokine production and neutrophil myeloperoxidase release were analyzed. Monocytes were isolated using anti-CD14 paramagnetic beads, and oligo (dT)25 beads were used to isolate mRNA before subsequent reverse transcription and semiquantitative amplification of various cytokines in order to determine time-related changes in expression during bypass. After 2 h, mRNAs for IL-1 beta and IL-6 were highly upregulated in noncoated compared to heparin-coated circuits. Little or no change was seen in the expression of other cytokines. IL-1 beta and IL-6 were measured in plasma after 12 h and reflected the upregulated mRNAs in noncoated circuits. A significantly reduced release of myeloperoxidase was observed in coated versus noncoated circuits. This indicates that heparin-coated surfaces reduce cellular activation and the release of inflammatory mediators.

Ability of flt3 ligand to stimulate the in vitro growth of primitive murine hematopoietic progenitors is potently and directly inhibited by transforming growth factor-beta and tumor necrosis factor-alpha.

The recently cloned flt3 ligand (FL) stimulates the growth of primitive hematopoietic progenitor cells through synergistic interactions with multiple other cytokines. The present study is the first demonstrating cytokines capable of inhibiting FL-stimulated hematopoietic cell growth. Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta l) potently inhibited the clonal growth of murine Lin-Sca-l+ bone marrow progenitors stimulated by FL alone or in combination with granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), interleukin (IL)-3, IL-6, IL-11, or IL-12. TGF-beta 1 inhibited more than 96% of the myeloid colony formation in response to these cytokine combinations, whereas TNF-alpha reduced the number of colonies by 58% to 96% depending on the cytokine by which FL was combined. In addition, both TNF-alpha and TGF-beta 1 inhibited more than 90% of B220+ cell production from B220- bone marrow cells stimulated by FL + IL-7. The effects of TNF-alpha and TGF-beta 1 appeared to be due to a direct effect and on the early progenitors because the inhibition was observed at the single cell level, and because delayed addition of the two inhibitors for only 48 hours dramatically reduced their inhibitory effects. A neutralizing anti-TGF-beta antibody showed the presence of endogenous TGF-beta in the cultures and potently enhanced the ability of FL to stimulate progenitor cell growth in the absence of other cytokines. Agonistic antibodies specifically activating the p75 TNF receptors were more efficient than wild type murine TNF-alpha in signaling growth inhibition of Lin-Sca-l+ progenitor cells, whereas the p55 agonist had less effect than murine TNF-alpha. Finally, TGF-beta increased the number of FL + IL-11-stimulated Lin-Sca-1+ cells in the G1 phase of the cell cycle with 76%, whereas TNF-alpha only had a marginal effect on cell cycle distribution. Thus, TGF-beta, TNF-alpha, and p75 TNF receptor agonists are potent direct inhibitors of FL-stimulated progenitor cell growth in vitro.

Cytokine modulation in experimental endotoxemia: characterization of an ex vivo whole blood model.

A new model was developed to study cytokine regulation and modulation in whole blood ex vivo. The model is characterized by stable leukocyte counts and high leukocyte viability throughout the experimental period. Oxygen consumption per time decreased slowly, whereas carbon dioxide partial pressure increased accordingly throughout the experiment. In this model, the anti-inflammatory effects of recombinant human (rh) interleukin (IL)-4, rhIL-10 and rhIL-13 on lipopolysaccharide (LPS) stimulated (10 ng/ml) leukocytes were examined and compared by measuring their ability to inhibit the release and mRNA levels of tumor necrosis factor (TNF)alpha, IL-6 and IL-1beta. rhIL-10 potently inhibited the release of TNF-alpha, IL-6 and IL-1beta in a potent and dose-dependent manner, but did not influence the mRNA levels of these cytokines in CD14-positive cells. Also, rhIL-4 and rhIL-13 inhibited the release of IL-6 and IL-1beta in a potent and dose-dependent manner, however, stronger maximal inhibition of IL-1beta (85%) than of IL-6 (60%) was obtained. In contrast, rhIL-4 and rhIL-13 seemed to have both stimulatory and inhibitory effects on plasma values of TNF-alpha. The effects of 10 ng/ml LPS showed to be signalling through the CD14 receptor, since blood treated with a monoclonal anti-CD14 antibody did not produce any TNF-alpha. The whole blood model described in this study is in our opinion a useful tool for investigating immunomodulating effects on a mixed white blood cell population.

Sirolimus interferes with the innate response to bacterial products in human whole blood by attenuation of IL-10 production.

Current immunosuppressive strategies are aimed at abrogating the allospecific T-cell response against donor tissues or organs. However, little information is yet available on the potential influences of these drugs on innate immune responses. In order to address this, we have employed a whole blood model. Human whole blood was pretreated with sirolimus, cyclosporine A or tacrolimus in therapeutic as well as supra therapeutic doses, and subsequently stimulated with lipopolysaccharide (LPS), peptidoglycan (PepG) or lipoteichoic acid (LTA). Plasma cytokine analyses revealed a potent inhibitory effect of sirolimus on interleukin(IL)-10 production induced by all bacterial products tested. In contrast, cyclosporine A and tacrolimus inhibited the tumour necrosis factor (TNF)-alpha production in response to LPS, but not to PepG and LTA. Using a quantitative mRNA analyses, we also observed that sirolimus significantly decreased the IL-10 mRNA accumulation to sub-basal levels in peripheral blood mononuclear cells (PBMC). This suggests that the sirolimus inhibits IL-10 production by interfering with the IL-10 gene transcription. However, the molecular mechanism of this inhibition remains unclear. Based on the present study and observations by others, we postulate that the clinical use of the sirolimus may be associated with a dysregulated innate immune response to bacterial infection and thus an increased risk of hyperinflammation and sepsis.

Veno-venous bypass in liver transplantation: heparin-coated perfusion circuits reduce the activation of humoral defense systems in an in vitro model.

We studied the effects of bypass circuit surface heparinization on kallikrein-kinin, coagulation, fibrinolytic and complement activation in a closed model system for simulating veno-venous bypass (WBP) in orthotopic liver transplantation (OLT). The circuits were identical to those in routine use during clinical OLT in our institution. Fresh whole human blood diluted 1:2 with Ringer's acetate was circulated at a non-pulsatile flow (2 l/min) and at a constant temperature (37.5 degrees C) for 12 h. In 10 experiments, the entire inner surface of the circuits was coated with end-point attached heparin (HC). In the remaining 10, non-treated PVC tubing was used (NC). Components of the plasma kallikrein-kinin, coagulation, fibrinolytic and complement systems were analyzed using functional techniques (chromogenic peptide substrate assays) and enzyme immunoassays at baseline, 3 and 12 h. Significant activation of the initial (C3bc) and terminal (TCC) components of the complement system were found in both the NC and HC groups after 3 and 12 h: C3bc: NC: baseline = 4 (3.5-7.7), 3 h = 17.3* (12.5-27), 12h = 31* (17.7-63.6), HC: baseline = 4.9 (3.2-6.8), 3h = 9* (6-14.4), 12h = 13.7* (7.4-18.1). TCC: NC: baseline = 0.4 (0.2-0.6), 3h = 5*(0.8-11.9), 12 h: 13.1* (4.2-25.7). HC: baseline = 0.5 (0.1-0.6), 3 h = 0.6* (0.1-0.8), 12 h = 1.2* (0.3-2) AU/ml; median and range (*: p < 0.05). The C3bc and TCC concentrations were significantly higher in the NC group at 3 and 12 h, compared to the HC group: C3bc (NC vs. HC group): 3 h, p < 0.001; 12 h, p < 0.001. TCC (NC vs. HC group): 3h, p < 0.001; 12 h, p < 0.001. Significant increases in the values of thrombin-antithrombin complexes (p = 0.003), prothrombin fragment 1 + 2 (p = 0.006) and plasmin-alpha2-antiplasmin complexes (p = 0.016) were found in the non-coated group, but not in the heparin-coated group during the observation period, showing that the coagulation and fibrinolytic systems were activated in the non-coated circuits. We conclude that heparin-coating of the internal surface of the extracorporeal perfusion circuit used for WBP reduces activation of the plasma cascade systems in a closed venous system in vitro.