Pavle Milenković

Interleukin-6 (IL-6) and low O2 concentration (1%) synergize to improve the maintenance of hematopoietic stem cells (pre-CFC)

Low O2 concentration (1%) favors the self‐renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL‐6 influences both stem cells and committed progenitors at 20% O2, we studied its effects in cultures at 1% O2. The pre‐CFC activity in Lin− population of mouse bone marrow was analyzed following 10 days of serum‐free culture in medium (LC1) supplemented with IL‐3 with and without IL‐6, at 20 and 1% O2 and phenotypic differentiation and proliferative history monitored. The IL‐6 receptor expression and initiation of VEGF‐A synthesis were also investigated. At 20% O2, the effects of IL‐6 on pre‐CFC were negligible but effects on CFC were apparent; conversely, at 1% O2, the IL‐6 enhances activity of pre‐CFC but not of CFC. Unlike at 20% O2, at 1% O2 a subpopulation of cells remained Lin− in spite of extensive proliferation. However, the absolute number of Lin− cells, did not correlate with pre‐CFC activity. A relative increase in VEGF transcripts at 1% O2 in presence of IL‐3 alone was enhanced by the addition of IL‐6. IL‐6 enhanced pre‐CFC activity at 1% O2 and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O2 does not exist in tissues in vivo, further studies in vitro at lower O2 concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells. J. Cell. Physiol. 212: 68–75, 2007.

The effect of a plasma needle on bacteria in planktonic samples and on peripheral blood mesenchymal stem cells

In this paper, we study the application of a plasma needle to induce necrosis in planktonic samples containing a single breed of bacteria. Two different types of bacteria, Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), were covered in this study. In all experiments with bacteria, the samples were liquid suspensions of several different concentrations of bacteria prepared according to the McFarland standard. The second system studied in this paper was human peripheral blood mesenchymal stem cells (hPB-MSC). In the case of hPB-MSC, two sets of experiments were performed: when cells were covered with a certain amount of liquid (indirect) and when the cell sample was in direct contact with the plasma.Most importantly, the study is made with the aim to see the effects when the living cells are in a liquid medium, which normally acts as protection against the many agents that may be released by plasmas. It was found that a good effect may be expected for a wide range of initial cell densities and operating conditions causing destruction of several orders of magnitude even under the protection of a liquid. It was established independently that a temperature increase could not affect the cells under the conditions of our experiment, so the effect could originate only from the active species produced by the plasma. In the case of those hPB-MSC that were not protected by a liquid, gas flow proved to produce a considerable effect, presumably due to poor adhesion of the cells, but in a liquid the effect was only due to the plasma. Further optimization of the operation may be attempted, opening up the possibility of localized in vivo sterilization.

In vivo effects of interleukin-17 on haematopoietic cells and cytokine release in normal mice

Abstract.  In order to gain more insight into mechanisms operating on the haematopoietic activity of the T‐cell‐derived cytokine, interleukin‐17 (IL‐17) and target cells that first respond to its action in vivo, the influence of a single intravenous injection of recombinant mouse IL‐17 on bone marrow progenitors, further morphologically recognizable cells and peripheral blood cells was assessed in normal mice up to 72 h after treatment. Simultaneously, the release of IL‐6, IL‐10, IGF‐I, IFN‐γ and NO by bone marrow cells was determined. Results showed that, in bone marrow, IL‐17 did not affect granulocyte‐macrophage (CFU‐GM) progenitors, but induced a persistant increase in the number of morphologically recognizable proliferative granulocytes (PG) up to 48 h after treatment. The number of immature erythroid (BFU‐E) progenitors was increased at 48 h, while the number of mature erythroid (CFU‐E) progenitors was decreased up to 48 h. In peripheral blood, white blood cells were increased 6 h after treatment, mainly because of the increase in the number of lymphocytes. IL‐17 also increased IL‐6 release and NO production 6 h after administration. Additional in vitro assessment on bone marrow highly enriched Lin− progenitor cells, demonstrated a slightly enhancing effect of IL‐17 on CFU‐GM and no influence on BFU‐E, suggesting the importance of bone marrow accessory cells and secondary induced cytokines for IL‐17 mediated effects on progenitor cells. Taken together, these results demonstrate that in vivo IL‐17 affects both granulocytic and erythroid lineages, with more mature haematopoietic progenitors responding first to its action. The opposite effects exerted on PG and CFU‐E found at the same time indicate that IL‐17, as a component of a regulatory network, is able to intervene in mechanisms that shift haematopoiesis from the erythroid to the granulocytic lineage.

Effect of IL-17 on In Vitro Hematopoietic Progenitor Cells Growth and Cytokine Release in Normal and Post-irradiated Murine Bone Marrow

Abstract The influence of recombinant human IL-17 on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) progenitors and the release of IL-1α/β, IL-6 and erythropoietin (EPO) was estimated in the bone marrow cells obtained from normal and sub-lethally irradiated mice. In normal mice IL-17 increased CFU-GM and BFU-E and reduced CFU-E derived colonies numbers and augmented release of IL-6 and EPO. In irradiated mice the effects of IL-17 on hematopoietic progenitors were lineage-dependent, as well as dependent on their stage of differentiation and the time after the irradiation. IL-17 had no major effects on CFU-GM on day 1 and 3, but decreased their number on day 2, while enhanced both BFU-E and CFU-E on day 1 and 2 after irradiation, whereas on day 3 its effect on erythroid progenitors was again as observed in normal mice. After irradiation, IL-17 increased the release of IL-1α, IL-6 and EPO. The observed effects suggested the involvement of IL-17 in the regulation of hematopoiesis and indicated that its effects on both hematopoietic progenitors and cytokine release are dependent on the physiological/ pathological status of the organism.

Signaling Pathways Implicated in Hematopoietic Progenitor Cell Proliferation and Differentiation

The objective of this study was to investigate the signal transduction pathways associated with the clonal development of myeloid and erythroid progenitor cells. The contribution of particular signaling molecules of protein tyrosine kinases (PTKs), mitogen-activated protein (MAP) kinase, and PI-3 kinase signaling to the growth of murine bone marrow colony forming unit–granulocyte-macrophage (CFU-GM) and erythroid (burst forming unit-erythroid [BFU-E] and colony forming unit-erythroid [CFU-E]) progenitors was examined in studies performed in the presence or absence of specific signal transduction inhibitors. The results clearly pointed to different signal transducing intermediates that are involved in cell proliferation and differentiation depending on the cell lineage, as well as on the progenitors’ maturity. Lineage-specific differences were obtained when chemical inhibitors specific for receptor- or nonreceptor-PTKs, as well as for the main groups of distinctly regulated MAPK cascades, were used because all of these compounds suppressed the growth of erythroid progenitors, with no major effects on myeloid progenitors. At the same time, differential involvement of MEK/extracellular signal-regulated kinase (ERK) MAPK transduction pathway was observed in the proliferation and/or differentiation of early, BFU-E, and late, CFU-E, erythroid progenitor cells. The results also demonstrated that phosphatydylinositol (PI)-3 kinase and nuclear factor kappaB (NF-κB) transcriptional factor were required for maintenance of both myeloid and erythroid progenitor cell function. Overall, the data obtained indicated that committed hematopoietic progenitors express a certain level of constitutive signaling activity that participates in the regulation of normal steady-state hematopoiesis and point to the importance of evaluating the impact of signal transduction inhibitors on normal bone marrow when used as potential therapeutic agents.

Low O 2 concentrations enhance the positive effect of IL-17 on the maintenance of erythroid progenitors during co-culture of CD34+ and mesenchymal stem cells

Co-culture of haematopoietic cells with a stromal cell layer does not mimic the physiological, micro-environmental niche, whose major feature is a low oxygen (O2) concentration. Thus, in order to study the effects of IL-17 in a context which better approximates the physiological state, we investigated its effects on cell expansion, colony-forming ability, and the phenotypical profile of normal, human blood CD34+ cells co-cultured for five days with MSC layers at various O2 concentrations (20%, 12.5% and 3% O2. We demonstrated that IL-17 enhances CD34+ and total CFC production during the five days of MSC/CD34+ co-culture. This effect depends upon the O2 concentration, reaching its maximum at 3% O2, and is more pronounced on erythroid progenitors (BFU-E). In addition, the stimulation of IL-6 production by IL-17 in MSC cultures and co-cultures is enhanced by low O2 concentration. The expression of some differentiation markers (CD34, CD13 and CD41) on haematopoietic cells in co-cultures also depends upon the oxygen concentration. Our results strengthen the concept that physiological levels of O2 (mistakenly called hypoxia), should be considered as an important environmental factor that significantly influences cytokine activity.

p38 MAPK signaling mediates IL-17-induced nitric oxide synthase expression in bone marrow cells

The effects of interleukin (IL)-17 on nitric oxide (NO) synthase (NOS) expression, as well as the participation of mitogen-activated protein kinases (MAPKs) in IL-17-mediated effects were examined in murine bone marrow cells. The results demonstrated the ability of IL-17 to upregulate the expression of mRNA for both inducible NOS and constitutive, endothelial NOS isoforms, as well as to enhance the phosphorylation of p38 MAPK. Moreover, both the NOS-inducing effect of IL-17 and the in vitro IL-17-mediated inhibition colony forming unit-erythroid (CFU-E) growth were dependent on p38 MAPK activity. The data demonstrating that the in vivo reducing effect of IL-17 on bone marrow CFU-E was prevented by co-treatment with the NOS inhibitor Nw-nitro-l-arginine methyl ester hydrochloride (L-NAME), implied that this effect is mediated through NOS activation. Besides revealing a link between the IL-17, NO, and haematopoiesis, data presented gave an insight into the mechanisms by which IL-17 exerts its modulatory effects on bone marrow cells.

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34+ cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34+ cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

Combined effect of IL-17 and blockade of nitric oxide biosynthesis on haematopoiesis in mice

Aim:  The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)‐17 and the specific role of nitric oxide (NO) in IL‐17‐induced effects in the process of haematopoiesis.

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

Abstract. 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.