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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

Effects of LPS alone or with IL-6 on CD41 expression and polyploidization.(A) Bone marrow cells were grown in a liquid serum-free medium supplemented with LPS or IL-6 alone or together for 3 d. Megakaryocytes were stained with FITC-conjugated anti-mouse CD41 monoclonal antibody and analyzed by FACS with large forward-scatter and side-scatter properties. The experiment was repeated three times, and similar results were obtained. (B) Bone marrow cells from TPO-pretreated mice were isolated on a discontinuous BSA density gradient and grown in a liquid serum-free medium in the presence of IL-6 or LPS alone or together for 3 d. The flow cytometric analysis of the DNA content of CD41-positive cells was examined by PI staining. The experiment was repeated three times, and similar results were obtained. (C) The histogram plots of PI staining were calculated from the percentage of DNA content.
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f4: Effects of LPS alone or with IL-6 on CD41 expression and polyploidization.(A) Bone marrow cells were grown in a liquid serum-free medium supplemented with LPS or IL-6 alone or together for 3 d. Megakaryocytes were stained with FITC-conjugated anti-mouse CD41 monoclonal antibody and analyzed by FACS with large forward-scatter and side-scatter properties. The experiment was repeated three times, and similar results were obtained. (B) Bone marrow cells from TPO-pretreated mice were isolated on a discontinuous BSA density gradient and grown in a liquid serum-free medium in the presence of IL-6 or LPS alone or together for 3 d. The flow cytometric analysis of the DNA content of CD41-positive cells was examined by PI staining. The experiment was repeated three times, and similar results were obtained. (C) The histogram plots of PI staining were calculated from the percentage of DNA content.

Mentions: To determine the effect of LPS and IL-6 on the terminal differentiation of megakaryocytes, the cells were also analyzed to investigate CD41 expression by flow cytometry. Low-density bone marrow cells from normal mouse cells were incubated either with 50.0 ng/ml IL-6 or with IL-6 combined with different doses of LPS for 3 d. The results of direct FITC fluorescence staining and flow cytometric analysis of CD41 indicated the percentage of cells undergoing megakaryopoiesis. Figure 4A shows that only 27.50 ± 3.3% and 42.59 ± 7.1% of the cells were CD41 positive in the presence of LPS (10.0 ng/ml) and IL-6 (50.0 ng/ml), respectively. However, the CD41-positive cells showed significant changes in the cultures treated with LPS and IL-6. The number of CD41-positive cells in the IL-6 (50.0 ng/ml) plus LPS-supplemented (10.0 ng/ml) culture was higher (81.22 ± 8.4%) than that observed in the IL-6 alone or IL-6 plus 1000.0 ng/ml LPS cultures (13.12 ± 5.6%, ). We also examined the nuclear DNA content of the CD41-positive cells stained with PI to determine the polyploidization of the treated cells. Figure 4B shows that a significant populations of the CD41-positive cells were 4N (49.25 ± 3.3%) and 4N (45.12 ± 2.5%) after the bone marrow cells were treated with alone LPS or IL-6. However, the megakaryocyte ploidy number was either 4N (22.81 ± 4.5%) or 8N (13.54 ± 2.1%) in the IL-6 plus 10.0 ng/ml LPS culture, with 16N (8.52 ± 3.7%) megakaryocytes being also observed. Conversely, a significantly decreased population of megakaryocytes was produced in IL-6-treated cultures with 1000.0 ng/ml LPS; these megakaryocytes contained a DNA content of 2N and 4N (54.87 ± 2.6% and 4.23 ± 1.8%, respectively). It should be noted that a significant population of makaryocytes produced in IL-6 plus LPS (10.0 ng/ml)-contained cultures had relative higher counts in term of 4N DNA content compared with the other treatments, which was coupled with an increase in the population of cells in 8N DNA content (Fig. 4C).


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)

Effects of LPS alone or with IL-6 on CD41 expression and polyploidization.(A) Bone marrow cells were grown in a liquid serum-free medium supplemented with LPS or IL-6 alone or together for 3 d. Megakaryocytes were stained with FITC-conjugated anti-mouse CD41 monoclonal antibody and analyzed by FACS with large forward-scatter and side-scatter properties. The experiment was repeated three times, and similar results were obtained. (B) Bone marrow cells from TPO-pretreated mice were isolated on a discontinuous BSA density gradient and grown in a liquid serum-free medium in the presence of IL-6 or LPS alone or together for 3 d. The flow cytometric analysis of the DNA content of CD41-positive cells was examined by PI staining. The experiment was repeated three times, and similar results were obtained. (C) The histogram plots of PI staining were calculated from the percentage of DNA content.
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Related In: Results  -  Collection

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f4: Effects of LPS alone or with IL-6 on CD41 expression and polyploidization.(A) Bone marrow cells were grown in a liquid serum-free medium supplemented with LPS or IL-6 alone or together for 3 d. Megakaryocytes were stained with FITC-conjugated anti-mouse CD41 monoclonal antibody and analyzed by FACS with large forward-scatter and side-scatter properties. The experiment was repeated three times, and similar results were obtained. (B) Bone marrow cells from TPO-pretreated mice were isolated on a discontinuous BSA density gradient and grown in a liquid serum-free medium in the presence of IL-6 or LPS alone or together for 3 d. The flow cytometric analysis of the DNA content of CD41-positive cells was examined by PI staining. The experiment was repeated three times, and similar results were obtained. (C) The histogram plots of PI staining were calculated from the percentage of DNA content.
Mentions: To determine the effect of LPS and IL-6 on the terminal differentiation of megakaryocytes, the cells were also analyzed to investigate CD41 expression by flow cytometry. Low-density bone marrow cells from normal mouse cells were incubated either with 50.0 ng/ml IL-6 or with IL-6 combined with different doses of LPS for 3 d. The results of direct FITC fluorescence staining and flow cytometric analysis of CD41 indicated the percentage of cells undergoing megakaryopoiesis. Figure 4A shows that only 27.50 ± 3.3% and 42.59 ± 7.1% of the cells were CD41 positive in the presence of LPS (10.0 ng/ml) and IL-6 (50.0 ng/ml), respectively. However, the CD41-positive cells showed significant changes in the cultures treated with LPS and IL-6. The number of CD41-positive cells in the IL-6 (50.0 ng/ml) plus LPS-supplemented (10.0 ng/ml) culture was higher (81.22 ± 8.4%) than that observed in the IL-6 alone or IL-6 plus 1000.0 ng/ml LPS cultures (13.12 ± 5.6%, ). We also examined the nuclear DNA content of the CD41-positive cells stained with PI to determine the polyploidization of the treated cells. Figure 4B shows that a significant populations of the CD41-positive cells were 4N (49.25 ± 3.3%) and 4N (45.12 ± 2.5%) after the bone marrow cells were treated with alone LPS or IL-6. However, the megakaryocyte ploidy number was either 4N (22.81 ± 4.5%) or 8N (13.54 ± 2.1%) in the IL-6 plus 10.0 ng/ml LPS culture, with 16N (8.52 ± 3.7%) megakaryocytes being also observed. Conversely, a significantly decreased population of megakaryocytes was produced in IL-6-treated cultures with 1000.0 ng/ml LPS; these megakaryocytes contained a DNA content of 2N and 4N (54.87 ± 2.6% and 4.23 ± 1.8%, respectively). It should be noted that a significant population of makaryocytes produced in IL-6 plus LPS (10.0 ng/ml)-contained cultures had relative higher counts in term of 4N DNA content compared with the other treatments, which was coupled with an increase in the population of cells in 8N DNA content (Fig. 4C).

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