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Micro-concentration Lipopolysaccharide as a Novel Stimulator of Megakaryocytopoiesis that Synergizes with IL-6 for Platelet Production.

Wu D, Xie J, Wang X, Zou B, Yu Y, Jing T, Zhang S, Zhang Q - Sci Rep (2015)

Bottom Line: We found that serial doses of micro-concentration LPS significantly increased the platelet count in mice treated with kanamycin, along with increased expression of IL-6 compared with IL-3 and TPO in megakaryocytes obtained from the mouse bone morrow following LPS administration.Furthermore, LPS at lower levels ranging plus IL-6 effectively stimulated CFU-MK formation and increased CD41 expression and megakaryocyte polyploidization.Notably, the optimal LPS concentration in combination with IL-6 might be a novel stimulator of TLR4 and IL-6R expression in Dami cell lines, which initially occurs through TLR4-IL-6R crosstalk and then involves the activation of NF-κB and phosphorylation of p38 MAPK.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.

ABSTRACT
Lipopolysaccharide (LPS) induces platelet activation and enhances platelet sensitivity to aggregation, which might alter platelet counts. We found that serial doses of micro-concentration LPS significantly increased the platelet count in mice treated with kanamycin, along with increased expression of IL-6 compared with IL-3 and TPO in megakaryocytes obtained from the mouse bone morrow following LPS administration. Furthermore, LPS at lower levels ranging plus IL-6 effectively stimulated CFU-MK formation and increased CD41 expression and megakaryocyte polyploidization. Meanwhile, there was a sustained rise in the percentage of reticulated platelets in the whole blood in response to low-dosage LPS combined with IL-6. In vivo experiments also demonstrated that the administration of LPS combined with IL-6 substantially enhanced the number of circulating platelets in normal and thrombocytopenic mice. Notably, the optimal LPS concentration in combination with IL-6 might be a novel stimulator of TLR4 and IL-6R expression in Dami cell lines, which initially occurs through TLR4-IL-6R crosstalk and then involves the activation of NF-κB and phosphorylation of p38 MAPK. These data suggest a new paradigm for the regulation of megakaryocytopoiesis and platelet production via a synergistic effect of LPS and IL-6, which has the potential to be used for the design of new therapies.

No MeSH data available.


Related in: MedlinePlus

Effect of LPS alone or with IL-6 on CFU-MKs.(A) Number of CFU-MKs in different mouse groups after incubation with LPS alone or IL-6 with LPS. Colonies classified according to their size were evaluated by inverted light microscopy. Experiments were performed in triplicate in four assays. Significant results compared with PBS are based on *P < 0.05 or **P < 0.01. (B) Bone marrow cells from TPO-pretreated mice were isolated by a discontinuous BSA density gradient and grown in a liquid serum-free medium in LPS alone or with IL-6 for 3 d. Cells were stained for acetylcholinesterase activity and phase contrast morphology of megakaryocyte cells. Scale bar, 10 μm.
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f3: Effect of LPS alone or with IL-6 on CFU-MKs.(A) Number of CFU-MKs in different mouse groups after incubation with LPS alone or IL-6 with LPS. Colonies classified according to their size were evaluated by inverted light microscopy. Experiments were performed in triplicate in four assays. Significant results compared with PBS are based on *P < 0.05 or **P < 0.01. (B) Bone marrow cells from TPO-pretreated mice were isolated by a discontinuous BSA density gradient and grown in a liquid serum-free medium in LPS alone or with IL-6 for 3 d. Cells were stained for acetylcholinesterase activity and phase contrast morphology of megakaryocyte cells. Scale bar, 10 μm.

Mentions: The in vitro levels of CFU-MK in a serum-free, semi-solid culture of murine bone marrow with different LPS concentrations (0.1 ng/ml to 1000.0 ng/ml) and IL-6 (50.0 ng/ml) were tested to determine the appropriate doses. The plateau levels of the dose-response curves were obtained at 50 ng/ml for IL-6 and 0.1 ng/ml to 10 ng/ml for LPS (data not shown). The proportions of CFU-MKs, estimated from the colonies containing 2 to 20 cells, 21 to 50 cells, and >50 cells, increased when the cells were exposed to IL-6 alone (20 ng/ml) or IL-6 with LPS (0.1 and 10 ng/ml). The treatment containing 50.0 ng/ml of IL-6 and 1000.0 ng/ml of LPS markedly reduced the number of CFU-MKs compared with the treatment with IL-6 only or IL-6 plus 0.1 or 10.0 ng/ml LPS (Fig. 3A). The majority of the increased number of CFU-MKs in IL-6 plus LPS treated cells were 3-20 cells/colony, whereas the number of CFU-MKs (>50 cells/colony) reached their highest level in the presence of IL-6 plus LPS (0.1 ng/ml). We next examined the morphological differences between the different stimulants. The megakaryocytes stimulated with 50 ng/ml IL-6 plus 0.1 ng/ml LPS had a more heavily stained cytoplasm than those stimulated with IL-6 alone, LPS alone or IL-6 (50.0 ng/ml) plus 10.0 or 1000.0 ng/ml LPS (Fig. 3C,E,F). The greater cell size with a heavily stained cytoplasm was also observed in the cells treated with 50.0 ng/ml IL-6 plus 0.1 ng/ml LPS (Fig. 3D). There were no other larger colonies, and a significant difference was observed among the other groups in these media based on microscopy analysis (Fig. 3B,E,F).


Micro-concentration Lipopolysaccharide as a Novel Stimulator of Megakaryocytopoiesis that Synergizes with IL-6 for Platelet Production.

Wu D, Xie J, Wang X, Zou B, Yu Y, Jing T, Zhang S, Zhang Q - Sci Rep (2015)

Effect of LPS alone or with IL-6 on CFU-MKs.(A) Number of CFU-MKs in different mouse groups after incubation with LPS alone or IL-6 with LPS. Colonies classified according to their size were evaluated by inverted light microscopy. Experiments were performed in triplicate in four assays. Significant results compared with PBS are based on *P < 0.05 or **P < 0.01. (B) Bone marrow cells from TPO-pretreated mice were isolated by a discontinuous BSA density gradient and grown in a liquid serum-free medium in LPS alone or with IL-6 for 3 d. Cells were stained for acetylcholinesterase activity and phase contrast morphology of megakaryocyte cells. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4557119&req=5

f3: Effect of LPS alone or with IL-6 on CFU-MKs.(A) Number of CFU-MKs in different mouse groups after incubation with LPS alone or IL-6 with LPS. Colonies classified according to their size were evaluated by inverted light microscopy. Experiments were performed in triplicate in four assays. Significant results compared with PBS are based on *P < 0.05 or **P < 0.01. (B) Bone marrow cells from TPO-pretreated mice were isolated by a discontinuous BSA density gradient and grown in a liquid serum-free medium in LPS alone or with IL-6 for 3 d. Cells were stained for acetylcholinesterase activity and phase contrast morphology of megakaryocyte cells. Scale bar, 10 μm.
Mentions: The in vitro levels of CFU-MK in a serum-free, semi-solid culture of murine bone marrow with different LPS concentrations (0.1 ng/ml to 1000.0 ng/ml) and IL-6 (50.0 ng/ml) were tested to determine the appropriate doses. The plateau levels of the dose-response curves were obtained at 50 ng/ml for IL-6 and 0.1 ng/ml to 10 ng/ml for LPS (data not shown). The proportions of CFU-MKs, estimated from the colonies containing 2 to 20 cells, 21 to 50 cells, and >50 cells, increased when the cells were exposed to IL-6 alone (20 ng/ml) or IL-6 with LPS (0.1 and 10 ng/ml). The treatment containing 50.0 ng/ml of IL-6 and 1000.0 ng/ml of LPS markedly reduced the number of CFU-MKs compared with the treatment with IL-6 only or IL-6 plus 0.1 or 10.0 ng/ml LPS (Fig. 3A). The majority of the increased number of CFU-MKs in IL-6 plus LPS treated cells were 3-20 cells/colony, whereas the number of CFU-MKs (>50 cells/colony) reached their highest level in the presence of IL-6 plus LPS (0.1 ng/ml). We next examined the morphological differences between the different stimulants. The megakaryocytes stimulated with 50 ng/ml IL-6 plus 0.1 ng/ml LPS had a more heavily stained cytoplasm than those stimulated with IL-6 alone, LPS alone or IL-6 (50.0 ng/ml) plus 10.0 or 1000.0 ng/ml LPS (Fig. 3C,E,F). The greater cell size with a heavily stained cytoplasm was also observed in the cells treated with 50.0 ng/ml IL-6 plus 0.1 ng/ml LPS (Fig. 3D). There were no other larger colonies, and a significant difference was observed among the other groups in these media based on microscopy analysis (Fig. 3B,E,F).

Bottom Line: We found that serial doses of micro-concentration LPS significantly increased the platelet count in mice treated with kanamycin, along with increased expression of IL-6 compared with IL-3 and TPO in megakaryocytes obtained from the mouse bone morrow following LPS administration.Furthermore, LPS at lower levels ranging plus IL-6 effectively stimulated CFU-MK formation and increased CD41 expression and megakaryocyte polyploidization.Notably, the optimal LPS concentration in combination with IL-6 might be a novel stimulator of TLR4 and IL-6R expression in Dami cell lines, which initially occurs through TLR4-IL-6R crosstalk and then involves the activation of NF-κB and phosphorylation of p38 MAPK.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China.

ABSTRACT
Lipopolysaccharide (LPS) induces platelet activation and enhances platelet sensitivity to aggregation, which might alter platelet counts. We found that serial doses of micro-concentration LPS significantly increased the platelet count in mice treated with kanamycin, along with increased expression of IL-6 compared with IL-3 and TPO in megakaryocytes obtained from the mouse bone morrow following LPS administration. Furthermore, LPS at lower levels ranging plus IL-6 effectively stimulated CFU-MK formation and increased CD41 expression and megakaryocyte polyploidization. Meanwhile, there was a sustained rise in the percentage of reticulated platelets in the whole blood in response to low-dosage LPS combined with IL-6. In vivo experiments also demonstrated that the administration of LPS combined with IL-6 substantially enhanced the number of circulating platelets in normal and thrombocytopenic mice. Notably, the optimal LPS concentration in combination with IL-6 might be a novel stimulator of TLR4 and IL-6R expression in Dami cell lines, which initially occurs through TLR4-IL-6R crosstalk and then involves the activation of NF-κB and phosphorylation of p38 MAPK. These data suggest a new paradigm for the regulation of megakaryocytopoiesis and platelet production via a synergistic effect of LPS and IL-6, which has the potential to be used for the design of new therapies.

No MeSH data available.


Related in: MedlinePlus